(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
22 November 2001 (22.11.2001)
PCT
(10) International Publication Number
WO 01/87337 Al
(51) International Patent Classification 7 :
39/44
A61K 39/395, THOMASSEN-WOLF, Elisabeth [DE/DE] ; Klingstrasse
12/3,81369 Miinchen (DE) .
(21) International Application Number: PCT/US01/15625
(22) International Filing Date: 14 May 2001 (14.05.2001)
(25) Filing Language: English
(26) Publication Language: English
(30) Priority Data:
00110065.0
60/238,492
12 May 2000 (12.05.2000) EP
6 October 2000 (06.10.2000) US
(71) Applicants (for all designated States except US):
GPC BIOTECH AG [DE/DE]; Fraunhoferstrasse 20,
82152 Martinsried/Miinchen (DE). MORPHOSYS
AG [DE/DE]; Lena-Christ-Strasse 48, 82152 Martin-
sried/Miinchen (DE).
(74) Agents: VINCENT, Matthew, P. et al.; Ropes & Gray,
One International Place, Boston, MA 02110-2624 (US).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, IP, KE, KG, KP, KR, KZ, LC,
EK, ER, ES, IT, EU, EV, MA, MD, MG, MK, MN, MW,
MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
SL, TI, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA,
ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TI, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BI, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
(72) Inventors; and
(75) Inventors/Applicants (for US only): NAGY, Zoltan
[DE/US]; One Kendall Square, Bldg. 600, Cambridge,
MA 02139 (US). BRUNNER, Christoph [DE/DE];
Kreutweg 8, 83673 Bichl (DE). TESAR, Michael
[DE/DE]; Karolingerstrasse 26, 82362 Weilheim (DE).
Published:
— with international search report
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations " appearing at the begin-
ning of each regular issue of the PCT Gazette.
m
m (54) Title: HUMAN POLYPEPTIDES CAUSING OR LEADING TO THE KILLING OF CELLS INCLUDING LYMPHOID TU-
MOR CELLS
go
(57) Abstract: The present invention relates to polypeptide compositions which bind to cell surface epitopes and, in multivalent
forms, cause or lead to the killing of cells including lymphoid tumor cells, and in the case of monovalent forms, cause immunosup-
C~J pression or otherwise inhibit activation of lymphocytes. The invention further relates to nucleic acids encoding the polypeptides,
methods for the production of the polypeptides, methods for killing cells, methods for immunosuppressing a patient, pharmaceutical,
diagnostic and multivalent compositions and kits comprising the polypeptides and uses of the polypeptides.
WO 01/87337
PCT/US01/15625
Human polypeptides causing or leading to the killing of cells including
lymphoid tumor cells
Background of the Invention
5
Every mammalian species, which has been studied to date, carries a cluster of genes
coding for the so-called major histocompatibility complex (MHC). This tightly linked
cluster of genes code for surface antigens, which play a central role in the
development of both humoral and cell-mediated immune responses. In humans the
10 products coded for by the MHC are referred to as Human Leukocyte Antigens or HLA.
The MHC-genes are organized into regions encoding three classes of molecules,
class I to III.
Class I MHC molecules are 45 kD transmembrane glycoproteins, noncovalently
15 associated with another glycoprotein, the 12 kD beta-2 microglobulin (Brown et al.,
1993). The latter is not inserted into the cell membrane, and is encoded outside the
MHC. Human class I molecules are of three different isotypes, termed HLA-A, -B, and
-C, encoded in separate loci. The tissue expression of class I molecules is ubiquitous
and codominant. MHC class I molecules present peptide antigens necessary for the
20 activation of cytotoxic T-cells.
Class II MHC molecules are noncovalently associated heterodimers of two
transmembrane glycoproteins, the 35 kD a chain and the 28 kD p chain (Brown et al.,
1993). In humans, class II molecules occur as three different isotypes, termed human
25 leukocyte antigen DR (HLA-DR), HLA-DP and HLA-DQ. Polymorphism in DR is
restricted to the p chain, whereas both chains are polymorphic . in the DP and DQ
isotypes. Class II molecules are expressed codominantly, but in contrast to class I,
exhibit a restricted tissue distribution: they are present only on the surface of cells of
the immune system, for example dendritic cells, macrophages, B lymphocytes, and
30 activated T lymphocytes. They are also expressed on human adrenocortical cells in
the zona reticularis of normal adrenal glands and on granulosa-lutein cells in corpora
lutea of normal ovaries (Kahoury et al., 1990). Their major biological role is to bind
antigenic peptides and present them on the surface of antigen presenting cells (APC)
for recognition by CD4 helper T (Th) lymphocytes (Babbitt et al., 1985). MHC class II
l
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molecules can also be expressed on the surface of non-immune system cells, for
example, cells that express MHC class II molecules during a pathological
inflammatory response. These cells may include synovial cells, endothelial cells,
thyroid stromal cells and glial cells.
5
Class III MHC molecules are also associated with immune responses, but encode
somewhat different products. These include a number of soluble serum proteins,
enzymes and proteins like tumor necrosis factor or steroid 21 -hydroxylase enzymes.
In humans, class III molecules occur as three different isotypes, termed Ca, C2 and
10 Bf (Kuby, 1994).
Since Th cell activation is a crucial event of the initiation of virtually all immune
responses and is mediated through class II molecules, class II MHC offers itself as a
target for immunomodulation (Baxevanis et al., 1980; Rosenbaum et al., 1981;
15 Adorini et al., 1988). Besides peptide presentation, class II molecules can transduce
various signals that influence the physiology of APC. Such signals arise by the
interaction of multiple class II molecules with an antibody or with the antigen receptor
of Th cells (Vidovic et al., 1995a; Vidovic et al., 1995b), and can induce B cell
activation and immunoglobulin secretion (Cambier et al., 1991; Palacios et al., 1983),
20 cytokine production by monocytes (Palacios, 1985) as well as the up-regulation of co-
stimulatory (Nabavi et al., 1992) and cell adhesion molecules (Mourad et al., 1990).
There is also a set of observations suggesting that class II ligation, under certain
conditions, can lead to cell growth arrest or be cytotoxic. Ligation under these
25 conditions is the interaction of a polypeptide with a class II MHC molecule. There is
substantial contradiction about the latter effects and their possible mechanisms.
Certain authors claim that formation of a complex of class II molecules on B cells
leads to growth inhibition (Vaickus et al., 1989; Kabelitz et al., 1989), whereas
according to others class II complex formation results in cell death (Vidovic et al.,
30 1995a; Newell et al., 1993; Truman et al., 1994; Truman et al., 1997; Drenou et al.,
1999). In certain experimental systems, the phenomenon was observed with resting B
cells only (Newell et al., 1993), or in other systems with activated B cells only (Vidovic
et al., 1995a; Truman et al., 1994).
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Based on these observations, anti-class II monoclonal antibodies (mAbs) have been
envisaged for a number of years as therapeutic candidates. Indeed, this proposal has
been supported by the beneficial effect of mouse-derived anti-class !l mAbs in a
series of animal disease models (Waldor et al., 1983; Jonker et al., 1988; Stevens et
5 al., 1990; Smith et al., 1994; Vidovic & Torral, 1998; Vidovic & Laus, 2000).
Despite these early supporting data, to date no anti-MHC class II mAb of human
composition has been described that displays the desired cytotoxic and other
biological properties which may include affinity, efficiency of killing and selectivity.
10 Indeed, despite the relative ease by which mouse-derived mAbs may be derived,
work using mouse-derived mAbs has demonstrated the difficulty of obtaining an
antibody with the desired biological properties. For example, significant and not fully
understood differences were observed in the T cell inhibitory capacity of different
murine anti-class II mAbs (Naquet et al., 1983). Furthermore, the application of
15 certain mouse-derived mAbs in vivo was associated with unexpected side effects,
sometimes resulting in death of laboratory primates (Billing et al., 1983; Jonker et al.,
1991).
It is generally accepted that mouse-derived mAbs (including chimeric and so-called
20 'humanized' mAbs) carry an increased risk of generating an adverse immune
response (Human anti-murine antibody - HAMA) in patients compared to treatment
with a human mAb (for example, Vose et al, 2000; Kashmiri et al., 2001). This risk is
potentially increased when treating chronic diseases such as rheumatoid arthritis or
multiple sclerosis with any mouse-derived mAb or where regular treatment may be
25 required, for example in the treatment of certain cancers; prolonged exposure of the
human immune system to a non-human molecule often leads to the development of
an adverse immune reaction. Furthermore, it has proven very difficult to obtain
mouse-derived antibodies with the desired specificity or affinity to the desired antigen
(Pichla et al. 1997). Such observation may significantly reduce the overall therapeutic
30 effect or advantage provided by mouse-derived mAbs. Examples of disadvantages
for mouse-derived mAbs may include the following. First, mouse-derived mAbs may
be limited in the medical conditions or length of treatment for a condition for which
they are appropriate. Second, the dose rate for mouse-derived mAbs may need to be
relatively high in order to compensate for a relatively low affinity or therapeutic effect,
3
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hence making the dose not only more severe but potentially more immunogenic and
perhaps dangerous. Third, such restrictions in suitable treatment regimes and high-
dose rates requiring high production amounts may significantly add to the cost of
treatment and could mean that such a mouse-derived mAb be uneconomical to
5 develop as a commercial therapeutic. Finally, even if a mouse mAb could be
identified that displayed the desired specificity or affinity, often these desired features
are detrimentally affected during the 'humanization' or 'chimerization' procedures
necessary to reduce immunogenic potential (Slavin-Chiorini et al., 1997). Once a
mouse-derived mAb has been 'humanized' or chimerized, then it is very difficult to
1 0 optimize its specificity or affinity.
The art has sought over a number of years for anti-MHC class II mAbs of human
composition that show biological properties suitable for use in a pharmaceutical
composition for the treatment of humans. Workers in the field have practiced the
15 process steps of first identifying a mouse-derived mAb, and then modifying the
structure of this mAb with the aim of improving immunotolerance of this non-human
molecule for human patients (for further details, see Jones et al., 1986; Riechmann et
al., 1988; Presta, 1992). This modification is typically made using so-called
'humanization' procedures or by fabricating a human-mouse chimeric mAb. Other
20 workers have attempted to identify human antibodies that bind to human antigens
having desired properties within natural repertoires of human antibody diversity. For
example, by exploring the foetal-tolerance mechanism in pregnant women (Bonagura
et al., 1987) or by panning libraries of natural diversities of antibodies (Stausb0l-Gr0n
et al., 1996; Winter et al., 1994). However, to date no anti-MHC class II mAb of
25 human composition has been described that displays the desired biological properties
of cytotoxicity, selectivity, specificity, low immunogenicity and affinity.
For therapeutic purposes a polypeptide reacting with many or at least most of the
common allelic forms of a human class II MHC molecule would be desirable - e.g., to
30 enable its use in diverse patient populations. Moreover, the candidate polypeptide
should be cytotoxic to a wide range of lymphoid tumors, and preferably is cytotoxic by
way of a mechanism common to such a range of tumor cells. To allow for a wide
range of possible applications, the polypeptide desired should mediate its cytotoxic
effect without the dependence on further components of the immune system. For
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therapeutic purposes most patients receive for the treatment of e.g. cancer standard
chemo- or radiotherapy. Most of these treatments leave the patient
immunocompromised. Any additional treatment that relies on an intact immune
system is therefore likely to fail. The underlying problem is further demonstrated in
5 humans who suffer from a disease that destroys the immune system, e.g. HIV.
Opportunistic infections and malignant transformations are able to escape the
immune-surveillance and cause further complications.
Summary of the Invention
10
One aspect of the present invention relates to a composition including a polypeptide
comprising at least one antibody-based antigen-binding domain of human composition
with binding specificity for an antigen expressed on the surface of a human cell, wherein
treating cells expressing the antigen with a multivalent polypeptide having two or more of
15 said antigen binding domains causes or leads to killing of the cells in a manner where
neither cytotoxic entities nor immunological mechanisms are needed for killing. In
certain in preferred embodiments the antigen is an MHC antigen, preferably an MHO
class II antigen, such as DR/DP/DQ or DR. For instance, in certain preferred
embodiments, the subject compositions include a polypeptide comprising at least one
20 antibody-based antigen-binding domain which binds to human HLA DR with a Kd of 1f4.M,
100nM, 10nM or even 1nM or less.
Another aspect of the present invention provides a composition including a multivalent
polypeptide comprising a plurality of antibody-based antigen-binding domains of human
25 composition with binding specificity for human HLA DR. Treating cells expressing HLA
DR with the multivalent polypetide causes or leads to killing of the cell in a manner where
neither cytotoxic entities nor immunological mechanisms are needed for killing. In certain
preferred embodiments, the said antigen-binding domains individually bind to the human
HLA DR with a K d of 1|uM, 100nM, 10nM or even 1nM or less. In certain preferred
30 embodiments, the multivalent polypeptide has an EC 50 of 100 nM. 10nM or even 1nM or
less for killing activated lymphoid cells, transformed cells and/or lymphoid tumor cells.
Still another aspect of the present invention provides a composition including a
polypeptide comprising at least one antibody-based antigen-binding domain that binds to
35 human HLA DR with a K d of 1|iiM, 100nM, 10nM or even 1nM or less, the antigen-binding
5
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domain being isolated by a method which includes isolation of human VL and VH
domains from a recombinant antibody library by ability to bind to at least one epitope of
human HLA DR. Treating a cell expressing HLA DR with a multivalent polypeptide having
two or more of the antigen binding domains causes or leads to killing of the cells in a
5 manner where neither cytotoxic entities nor immunological mechanisms are needed for
killing. In certain embodiments, the method for isolating the antigen-binding domain
includes the further steps of:
a. generating a library of variants of at least one of the CDR1, CDR2 and
CDR3 sequences of one or both of the VL and VH domains, and
10 b. isolation of VL and VH domains from the library of variants by ability to
bind to human HLA DR with a K d of 1uM or less.
In certain preferred embodiments, the composition of the present invention can be
characterized as including multivalent polypeptides having an EC 50 for killing transformed
1 5 cells at least 5-fold lower than the EC 50 for killing normal cells, and even more preferably
at least 10-fold, 100-fold and even 1000-fold less than for killing normal cells.
In certain preferred embodiments, the composition of the present invention are
characterized as including multivalent polypeptides having an EC 50 for killing activated
20 cells at least 5-fold lower than the EC 50 for killing unactivated cells, and even more
preferably at least 10-folded, 100-fold and even 1000-fold less than for killing unactivated
cells.
In certain preferred embodiments, the composition of the present invention are
25 characterized as including multivalent polypeptides having an EC 50 of 50nM or less for
killing transformed cells, and even more preferably an EC 50 of less than 10nM, 1nM and
even 0.1 nM. In certain embodiments, the subject multivalent polypeptides have an EC 50
for killing killing activated lymphoid cells, transformed cells and/or lymphoid tumor cells of
1 0OnM, 1 0nM or even 1 nM or less.
30
In certain embodiments, the subject compositions including multivalent polypeptides
selectively kill activated lymphoid cells. For example, such multivalent forms of the
subject compositions can be used to kill activated lymphoid cells are lymphoid tumor
cells representing a disease selected from B cell non-Hodgkin lymphoma, B cell
35 lymphoma, B cell acute lymphoid leukemia, Burkitt lymphoma, Hodgkin lymphoma, hairy
cell leukemia, acute myeloid leukemia, T cell lymphoma, T cell non-Hodgkin lymphoma,
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chronic myeloid leukemia, chronic lymphoid leukemia, and multiple myeloid leukemia.
Exemplary activated lymphoid tumor cells which can be killed include Priess, GRANTA-
519, KARPAS-422, KARPAS-299, DOHH-2, SR-786, MHH-CALL-4, MN-60, BJAB, RAJI,
L-428, HDLM-2, HD-MY-Z, KM-H2, L1236, BONNA-12, HQ-1, NALM-1 , L-363, EOL-1,
5 LP-1, RPMI-8226, and MHH-PREB-1 cell lines. In certain preferred embodiments, the
subject compositions have an EC 50 of 100nM or less, and preferably less than 10nM or
even 1nM, for killing at least one of B cell lymphoma cells and T cell lymphoma cells
selected from the list of KARPAS-422, DOHH-2, SR-7, MHH-CALL-4, MN-60, HD-MY-Z,
NALM-1 and LP-1. In certain instances, to effect cell killing, the target cells may require
10 further activation or pre-activation, such as by by incubation with Lipopolysaccharide
(LPS, 10 ug/ml), Interferon-gamma (IFN-y, Roche, 40 ng/ml) and/or phyto-hemagglutinin
(PHA, 5 ug/ml) to name but a few.
In certain embodiments, the multivalent forms of the subject compositions can be used to
15 kill non-lymphoid cells that express MHC class II molecules.
Certain embodiments, one or more the antigen binding domains of the subject
compositions bind to the p-chain of HLA-DR, e.g., the antigen-binding domain binds to
the first domain of the B-chain of HLA-DR.
20
In certain other embodiments, one or more the antigen binding domains of the subject
compositions bind to the a-chain of HLA-DR, e.g., the antigen-binding domain binds to
the first domain of the a-chain of HLA-DR.
25 In certain preferred embodiments, the the antigen binding domain(s) of the subject
compositions bind to one or more HLA-DR types selected from the group consisting of
DR1-0101, DR2-15021, DR3-0301, DR4Dw4-0401, DR4Dw1 0-0402, DR4Dw14-0404,
DR6-1302, DR6-1401, DR8-8031, DR9-9012, DRW53-B4*0101 and DRW52-B3*0101.
In preferred embodiments, the the antigen binding domains of the subject compositions
30 provide broad-DR reactivity, that is, the antigen-binding domain(s) of a given composition
binds to epitopes on at least 5 different of said HLA-DR types. In certain embodiments,
the antigen binding domain(s) of a polypeptide(s) of the subject compositions binds to a
plurality of HLA-DR types as to bind to HLA DR expressing cells for at least 60 percent of
the human population, more preferably at least 75 percent, and even more preferably 85
35 percent of the human population.
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In certain embodiments, the antigen-binding domains of the subject compositions include
a combination of a VH domain and a VL domain, wherein said combination is found in
one of the clones taken from the list of MS-GPC-1 , MS-GPC-6, MS-GPC-8, MS-GPC-10,
5 MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10, MS-GPC-8-17, MS-GPC-8-18,
MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-19, MS-GPC-8-6-27, MS-GPC-8-6-45, MS-
GPC-8-6-13, MS-GPC-8-6-47, MS-GPC-8-1 0-57, MS-GPC-8-27-7, MS-GPC-8-27-10 and
MS-GPC-8-27-41 .
10 In certain embodiments, the antigen-binding domains of the subject compositions include
a combination of HuCAL VH2 and HuCAL VA1, wherein the VH CDR3, VL CDR1 And VL
CDR3 is found in one of the clones taken from the list of MS-GPC-1, MS-GPC-8, MS-
GPC-10, MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10, MS-GPC-8-17, MS-
GPC-8-18, MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-19, MS-GPC-8-6-27, MS-GPC-
15 8-6-45, MS-GPC-8-6-13, MS-GPC-8-6-47, MS-GPC-8-1 0-57, MS-GPC-8-27-7, MS-GPC-
8-27-10 and MS-GPC-8-27-41.
In a further preferred embodiment, the antigen-binding domain is modified compared to a
parental antigen-binding domain of the present invention by addition, deletion and/or
20 substitution of amino acid residues, while maintaining the properties according to the
present invention, or improving one or more of said properties, of said parental antigen-
binding domain. This may include, but is not limited to, the modification of a nucleic acid
sequence encoding a parental antigen-binding domain for cloning purposes, the
modification of CDR regions in order to improve or modify antigen-binding affinity and/or
25 specificity, including the exchange of one or more CDR sequences of a parental antigen-
binding domain by corresponding CDR sequences from one or more different antigen-
binding domains, and the addition of peptide sequences for detection and/or purification
purposes. It is well within the scope of one of ordinary skill in the art to identify positions
in a given parental antigen-binding domain where an addition, deletion and/or
30 substitution should occur, to design and pursue the approach to achieve said addition,
deletion and/or substitution, and to test or assay whether the modified antigen-binding
domain has maintained the properties of, or exhibits one or more improved properties
compared to, the parental antigen-binding domain. Furthermore, one of ordinary skill
would be able to design approaches where collections or libraries of modified antigen-
35 binding domains are designed, constructed and screened to identify one or more
modified antigen-binding domain which have maintained the properties, or exhibit one or
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more improved properties compared to the parental antigen-binding domain. In one
example, the first amino acid residue of a HuCAL VH domain comprised in any antigen-
binding domain or the present invention, which is either E or Q depending on the
expression construct, may be exchanged by Q or E, respectively. Preferred regions to
5 optimize an antigen-binding domain by designing, constructing and screening collections
or libraries of modified antigen-binding domains according to the present invention
comprise the CDR regions, and most preferably CDR3 of VH and VL, CDR1 of VL and
CDR2 of VH domains.
10 In certain embodiments, the antigen-binding domains includes a combination of HuCAL
VH2 and HuCAL VA1, wherein the VH CDR3 sequence is taken from the consensus
CDR3 sequence
nnnnRGnFDn
wherein each n independently represents any amino acid residue; and/or
15 wherein the VL CDR3 sequence is taken from the consensus CDR3 sequence
QSYDnnnn
wherein each n independently represents any amino acid residue. For instance, the VH
CDR3 sequence can be SPRYGAFDY and/or the VL CDR3 sequence can be
QSYDLIRH or QSYDMNVH.
20
In certain embodiments, the antigen-binding domains of the subject antigen-binding
domain competes for antigen binding with an antibody including a combination of HuCAL
VH2 and HuCAL VA1, wherein the VH CDR3 sequence is taken from the consensus
CDR3 sequence
25 nnnnRGnFDn
each n independently represents any amino acid residue; and/or
the VL CDR3 sequence is taken from the consensus CDR3 sequence
QSYDnnnn
each n independently represents any amino acid residue. For instance, the VH CDR3
30 sequence can be SPRYGAFDY and/or the VL CDR3 sequence can be QSYDLIRH or
QSYDMNVH.
In certain preferred embodiments, the antigen-binding domain includes a VL CDR1
sequence represented in the general formula
35 SGSnnNIGnNYVn
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wherein each n independently represents any amino acid residue. For instance, the
CDR1 sequence is SGSESNIGNNYVQ.
In preferred embodiments, the mechanism of killing by multivalent forms of the subject
5 compositions involves an innate pre-programmed process of said cell. For instance, the
killing is non-apoptotic. Killing by the subject compositions can be dependent on the
action of non-caspase proteases, and/or killing which cannot be inhibited by zVAD-fmk or
zDEVD-fmk.
10 In certain preferred embodiments, the antibody-based antigen-binding domain is part of a
multivalent polypeptide including at least a F(ab')2 antibody fragment or a mini-antibody
fragment.
In certain preferred embodiments, the antibody-based antigen-binding domain is part of a
1 5 multivalent polypeptide comprising at least two monovalent antibody fragments selected
from Fv, scFv, dsFv and Fab fragments, and further comprises a cross-linking moiety or
moieties.
In certain preferred embodiments, the antibody-based antigen-binding domain is part of a
20 multivalent polypeptide comprising at least one full antibody selected from the antibodies
of classes lgG1 , 2a, 2b, 3, 4, IgA, and IgM.
In certain preferred embodiments, the antibody-based antigen-binding domain is part of a
multivalent polypeptide is formed prior to binding to said cell.
25
In certain preferred embodiments, the antibody-based antigen-binding domain is part of a
multivalent polypeptide is formed after binding to said cell.
In certain preferred embodiments, the antigen binding sites are cross-linked to a polymer.
30
Another aspect of the present invention provides a nucleic acid comprising a coding
sequence for an antigen-binding domain, such as those antigen binding domains
described above, or a multivalent polypeptide thereof. For example, in certain
embodiments, the nucleic acid includes a coding sequence for a polypeptide
35 comprising at least one antibody-based antigen-binding domain of human
10
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composition with binding specificity for an antigen expressed on the surface of a
human cell, wherein treating cells expressing the antigen with a multivalent form of
the polypeptide causes or leads to killing of said cell in a manner where neither
cytotoxic entities nor immunological mechanisms are needed for killing. In certain
5 embodiments, the nucleic acid includes a coding sequence for a polypeptide comprising
at least one antibody-based antigen-binding domain which binds to at least one epitope
of human HLA DR with a K d of 1|nM, 100nM, 10nM or even 1nM or less.
In certain embodiments, the nucleic acid includes a coding sequence for a polypeptide
10 comprising a plurality of antibody-based antigen-binding domains of human composition
with binding specificity for human HLA DR, wherein treating a cell expressing HLA DR
with the multivalent polypeptide causes or leads to killing of the cell in a manner where
neither cytotoxic entities nor immunological mechanisms are needed for killing. In
preferred embodiments, the antigen-binding domains individually bind to epitopes on the
1 5 human HLA DR with a K d of 1 p.M, 1 0OnM, 1 0nM or even 1 nM or less.
In certain embodiments, the nucleic acid includes a coding sequence for a multivalent
polypeptide comprising a plurality of antibody-based antigen-binding domains of human
composition with binding specificity for human HLA DR, wherein treating a cell
20 expressing HLA DR with said multivalent polypeptide causes or leads to killing of said cell
in a manner where neither cytotoxic entities nor immunological mechanisms are needed
for said cell killing. Preferably, the multivalent polypeptide has an EC 50 for killing killing
activated lymphoid cells, transformed cells and/or lymphoid tumor cells of 100nM, 10nM
or even 1nM or less.
25
Another aspect of the invention provides a vector comprising the coding sequence of any
one of the subject nucleic acids, e.g., as described above, and a transcriptional
regulatory sequence operably linked thereto.
30 Still another aspect of the present invention provides a host cell harboring at least one
subject nucleic acids or the subject vector. Another aspect of the present invention
provides a method for the production of a multivalent composition that causes or leads to
killing of cells in a manner where neither cytotoxic entities nor immunological
mechanisms are needed to cause or lead to said killing comprising culturing the host
35 cells under conditions wherein the nucleic acid is expressed either as a polypeptide
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comprising a plurality of antigen binding domains or as a polypeptide comprising at least
one antigen binding domains which is subsequently treated to form a multivalent
composition.
5 Another aspect of the present invention provides forms of the subject polypeptide or
nucleic acid compositions, formulated in a pharmaceutically acceptable carrier and/or
diluent. The present invention specifically contemplates the use of such compositions for
preparing a pharmaceutical preparation for the treatment of animals, especially humans.
1 0 Such pharmaceutical compositions can be used for the treatment of conditions involving
unwanted cell proliferation, particularly the treatment of a disorder involving transformed
cells expressing MHC class II antigens. For instance, the formulations can be used for
the treatment of a disorder selected from B cell non-Hodgkin lymphoma, B cell
lymphoma, B cell acute lymphoid leukemia, Burkitt lymphoma, Hodgkin lymphoma, hairy
15 cell leukemia, acute myeloid leukemia, T cell lymphoma, T cell non-Hodgkin lymphoma,
chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloid leukemia and B
cell precursor leukemia.
Such pharmaceutical preparations can be used for the treatment of diseases involving
20 unwanted activation of immune cells, such as in the treatment of a disorder selected from
rheumatoid arthritis, juvenile arthritis, multiple sclerosis, Grave's disease, insulin-
dependent diabetes, narcolepsy, psoriasis, systemic lupus erythematosus, ankylosing
spondylitis, transplant rejection, graft vs. host disease, Hashimoto's disease, myasthenia
gravis, pemphigus vulgaris, glomerulonephritis, thyroiditis, pancreatitis, insulitis, primary
25 biliary cirrhosis, irritable bowel disease and Sjogren syndrome.
Another aspect of the present invention provides a diagnostic composition including the
polypeptide or nucleic acid compositions of the present invention. In certain
embodiments, the diagnostic composition includes a polypeptide composition and a
30 cross-linking moiety or moieties.
Still another aspect of the present invention provides a method for killing a cell
expressing an antigen on the surface of said cell comprising the step of contacting the
cell with a multivalent polypeptide composition of the subject invention.
35
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Another aspect of the invention provides a method to identify patients that can be treated
with a multivalent polypeptide composition, formulated in a pharmaceutically acceptable
carrier and/or diluent comprising the steps of
a. Isolating cells from a patient;
5 b. Contacting said cells with the composition; and
c. Measuring the degree of killing or immunosuppression of said
cells.
The present invention also provides a kit to identify patients that can be treated with a
10 • multivalent polypeptide composition of the present invention, formulated in a
pharmaceutically acceptable carrier and/or diluent comprising
a. a multivalent polypeptide composition; and
b. Means to measure the degree of killing or immunosuppression of
said cells.
15 In certain embodiments, the kit includes a multivalent polypeptide composition, and a
cross-linking moiety. In other embodiments, the kit includes
a. a multivalent polypeptide composition, and
b. a detectable moiety or moieties, and
c. reagents and/or solutions to effect and/or detect binding of (i) to an
20 antigen.
Another aspect of the present invention provides a cytotoxic composition comprising a
multivalent polypeptide composition operably linked to a cytotoxic agent.
25 Stil another aspect of the invention provides an immunogenic composition comprising a
multivalent polypeptide composition operablly linked to an immunogenic agent.
Another aspect of the present invention provides a method to kill a cell comprising
contacting the cell with a multivalent polypeptide composition operablly linked a cytotoxic
30 or immunogenic agent.
Another aspect of the invention provides a method for treating a human to reduce the
severity of disorder involving unwanted proliferation/activation of cells expressing the
human 3-chain of HLA DR, comprising administering to the patient a a multivalent
35 polypeptidepolypeptide of the present invention. In certain embodiments, the disorder
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involves unwanted proliferation/activation of lymphoid cells, e.g., selected from B cell
non-Hodgkin lymphoma, B cell lymphoma, B cell acute lymphoid leukemia, Burkitt
lymphoma, Hodgkin lymphoma, hairy cell leukemia, acute myeloid leukemia, T cell
lymphoma, T cell non-Hodgkin lymphoma, chronic myeloid leukemia, chronic lymphoid
5 leukemia, multiple myeloid leukemia and B cell precursor leukemia.
Another aspect of the invention provides a use of a multivalent polypeptide composition
operably linked a cytotoxic or immunogenic agent for preparing a pharmaceutical
preparation for the treatment of animals
10
According to a preferred embodiment, the polypeptide is directed to a lymphoid cell
or a non-lymphoid cell that expresses MHC class II molecules. The latter type of cells
occur for example at pathological sites of inflammation and/or autoimmune diseases,
e.g. synovial cells, endothelial cells, thyroid stromal cells and glial cells, or it may also
15 comprise genetically altered cells capable of expressing MHC class II molecules.
Preferably, the polypeptide is directed to lymphoid tumor cells. More preferred are
lymphoid tumor cells that represent a disease selected from B cell non-Hodgkin
lymphoma, B cell lymphoma, B cell acute lymphoid leukemia, Burkitt lymphoma,
20 Hodgkin lymphoma, hairy cell leukemia, acute myeloid leukemia and B cell precursor
leukemia. Most preferred are lymphoid tumor cells from a cell line taken from the list
of GRANTA-519, PRIESS, KARPAS-422, DOHH-2, MHH-CALL-4, MN-60, BJAB, L-
428, BONNA-12, EOL-1, MHH-PREB-1 and MHH-CALL-2 cell lines.
25 In certain embodiments, the polypeptide binds to at least one epitope in the alpha-
chain of an HLA-DR molecule. In such embodiments, the polypeptide preferably
binds to at least one epitope in the first domain of the alpha-chain of HLA-DR, the first
domain being the N-terminal domain of the chain. For instance, the polypeptide can
be selected to bind to at least one epitope within the alpha-helix ranging from Glu 55 to
30 Tyr 79 of the alpha-chain of HLA-DR.
In other embodiments, the polypeptide binds to at least one epitope in the beta-chain
of an HLA-DR molecule. Preferably, the polypeptide binds to at least one epitope in
the first domain of the beta-chain of HLA-DR, the first domain being the N-terminal
35 domain of the chain.
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In certain embodiments, the mechanism of killing a target cell induced by the
polypeptide involves an innate pre-programmed process of said cell. Preferably, the
polypeptide induces a killing mechanism, which is not an apoptotic cell death process.
5
In a preferred embodiment the polypeptide induces a killing mechanism which is
dependent on the action of proteases other than caspases, e.g., is a caspase-
independent mechanism.
10 In a further embodiment the multivalent composition comprises at least one full
antibody which is selected from classes IgG1, 2a, 2b, 3, 4, IgA, and IgM.
In a further embodiment the multivalent composition comprises at least one of a
F(ab') 2 antibody fragment or mini-antibody fragment.
15
In a preferred embodiment the multivalent composition comprises at least two
monovalent antibody fragments selected from Fv, scFv, dsFv and Fab fragments, and
further comprises a cross-linking moiety or moieties.
20 The present invention also provides a composition including a polypeptide comprising
at least one antibody-based antigen-binding domain with a binding specificity for
human HLA DR wherein binding of said polypeptide to said epitope causes or leads
to suppression of the immune response and wherein said antigen-binding domain
includes a combination of a VH domain and a VL domain, wherein said combination
25 is found in one of the clones taken from the list of MS-GPC-1, MS-GPC-6, MS-GPC-
8, MS-GPC-1 0, MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10, MS-GPC-
8-17, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-19, MS-GPC-8-6-
27, MS-GPC-8-6-45, MS-GPC-8-6-13, MS-GPC-8-6-47, MS-GPC-8-1 0-57, MS-GPC-
8-27-7, MS-GPC-8-27-10 and MS-GPC-8-27-41 .
30
Another immunosuppressive composition of the present invention includes a polypeptide
comprising at least one antibody-based antigen-binding domain with a binding specificity
for a human MHC class II antigen with a K d of 1pM, 100nM, 10nM or even 1nM or less,
wherein treating cells expressing MHC class II antigen with the polypeptide causes or
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leads to suppression of the immune response, e.g., preferably with an IC50 of 1p.M,
100nM, 10nM or even 1nM or less.
Another immunosuppressive composition of the present invention includes a polypeptide
5 comprising at least one antibody-based antigen-binding domain of human composition
with a binding specificity for a human MHC class II antigen with a K d of 1uM , 100nM,
10nM or even 1nM or less, the antigen-binding domain being isolated by a method which
includes isolation of human VL and VH domains from a recombinant antibody display
library by ability to bind to human MHC class II antigen, wherein treating cells that
10 express MHC class II with said polypeptide causes or leads to suppression of the
immune response.
The subject immunosuppressive compositions can be generated using the antigen-
binding domain isolated by the further steps of:
15 a. generating a library of mutations at least one of the CDR1, CDR2 and
CDR3 domains of one or both of the VL and VH domains, and
b. isolation of VL and VH domains from the library of variants by ability to
bind to human MHC class II antigen with a K d of 1uM or less.
20 In preferred embodiments, the antigen binding domains of the immunosuppressive
composition binds to HLA-DR, and preferably to the p-chain of HLA-DR, and even
more preferably to the first domain of the p-chain of HLA-DR.
In certain preferred embodiments, the immunosuppressive composition have an IC 50 for
25 suppressing the immune response of 1 uM, 100nM, 10nM or even 1nM or less.
In certain preferred embodiments, the immunosuppressive composition have an IC 50 for
inhibiting of IL-2 secretion of 1 uM, 100nM, 10nM or even 1nM or less.
30 In certain preferred embodiments, the immunosuppressive composition have an IC 50 for
inhibiting of T cell proliferation of 1 uM, 100nM, 10nM or even 1nM or less.
In certain preferred embodiments, the immunosuppressive composition have antigen-
binding domain that bind to an epitope of one or more HLA-DR types selected from the
35 group consisting of DR1-0101, DR2-15021, DR3-0301, DR4Dw4-0401, DR4Dw1 0-0402,
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DR4Dw1 4-0404, DR6-1302, DR6-1401, DR8-8031, DR9-9012, DRw53-B4*0101 and
DRw52-B3*0101, and in preferred embodiments, the antigen-binding domain binds to at
least 5 different of said HLA-DR types (e.g., are pan-DR)
5 In certain embodiments, the immunosuppressive composition have antigen-binding
domain includes a combination of a VH domain and a VL domain, wherein said
combination is found in one of the clones taken from the list of MS-GPC-1, MS-GPC-6,
MS-GPC-8, MS-GPC-1 0, MS-GPC-8-1 , MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10,
MS-GPC-8-17, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-19, MS-
10 GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-1 3, MS-GPC-8-6-47, MS-GPC-8-1 0-57,
MS-GPC-8-27-7, MS-GPC-8-27-1 0 and MS-GPC-8-27-41.
In certain embodiments, the immunosuppressive composition have antigen-binding
domain includes a combination of HuCAL VH2 and HuCAL VA1, wherein the VH CDR3
1 5 sequence is taken from the consensus CDR3 sequence
nnnnRGnFDn
wherein each n independently represents any amino acid residue; and
wherein the VL CDR3 sequence is taken from the consensus CDR3 sequence
QSYDnnnn
20 wherein each n independently represents any amino acid residue.
For instance, the VH CDR3 sequence is SPRYGAFDY and/or the VL CDR3 sequence is
QSYDLIRH or QSYDMNVH.
In certain embodiments, the immunosuppressive composition the antigen-binding
25 domain competes with antigen binding by an antibody having a VH CDR3 sequence
represented by the general formula
nnnnRGnFDn
wherein each n independently represents any amino acid residue; and
a VL CDR3 sequence represented by the general formula
30 QSYDnnnn
wherein each n independently represents any amino acid residue.
In certain embodiments, the immunosuppressive composition the antigen-binding
domain includes a VL CDR1 sequence represented in the general formula
35 SGSnnNIGnNYVn
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wherein each n independently represents any amino acid residue. For example, the
CDR1 sequence is SGSESNIGNNYVQ.
In certain embodiments, the subject immunosuppressive compositions suppress the
5 immune response by one or more of (a) down-regulation of expression of the antigen to
which the polypeptide binds; or (b) inhibiting of the interaction between said cell and other
cells, wherein said interaction would normally lead to an immune response.
Another aspect of the present invention provides nucleic acids which including a coding
10 sequence for an immunosuppressive polypeptide of the present invention. In certain
embodiments, the nucleic acid can be provided as part of a vector, e.g., including the
coding sequence and a transcriptional regulatory sequence operably linked thereto. The
nucleic acid and vectors of the present invention can be provided as part of a host cell,
e.g., which can be used to to produce an immunosuppressive composition.
15
Another aspect of the present invention provides a method for suppressing activation
and/or proliferation of a lymphocyte, comprising contacting the cell with an
immunosuppressive polypeptide of the present invention.
20 The present invention also provides a pharmaceutical preparation comprising the a
polypeptide including an antibody-based antigen-binding domain with a binding specificity
for a human MHC class II antigen with a K d of 1uM or less, e.g., in an amount sufficient
to suppress an immune response in an animal, inhibit IL-2 secretion in an animal, and/or
inhibit T cell proliferation in an animal.
25
Another aspect of the present invention relates to the use of a polypeptide including an
antibody-based antigen-binding domain with a binding specificity for a human MHC class
II antigen with a K d of 1uM or less, for the preparation of a pharmaceutical composition
for the treatment of animals, such as where said animals are human.
30
The subject immunosuppressive pharmaceutical preparations can be used for
suppressing IL-2 secretion by a cell of the immune system. For example, these
preparations can be administered to the patient in an effective amount to reduce the level
of immunological responsiveness in the patient.
35
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Still another aspect of the present invention provides a method for suppressing IL-2
secretion by a lymphocyte, comprising contacting the cell with an immunosuppressive
polypeptide of the present invention.
5 The subject method can be used for immunosuppressing a human, e.g., by administering
to the patient an effective amount of an immunosuppressive polypeptide of the present
invention to reduce the level of immunological responsiveness.
The invention further relates to a diagnostic composition containing at least one
10 polypeptide and/or nucleic acid according to the invention, optionally together with
further reagents, such as buffers, for performing the diagnosis.
In a preferred embodiment the diagnostic composition contains the polypeptide
according to the invention cross-linked by at least one moiety. Such moieties can be
15 for example antibodies recognizing an epitope present on the polypeptide such as the
FLAG peptide epitope (Hopp et al., 1988; Knappik and Pluckthun, 1994) or
bifunctional chemical compounds reacting with a nucleophilic amino acid side chain
as present in cysteine or lysine (King et al., 1994). Methods for cross-linking
polypeptides are well known to the practitioner of ordinary skill in the art.
20
A diagnostic composition containing at least one nucleic acid and/or variant thereof
according to the invention is also contemplated.
Furthermore, the present invention relates to a kit comprising at least one polypeptide
25 according to the present invention, and a cross-linking moiety.
Additionally, the present invention relates to a kit comprising (i) a polypeptide
according to the present invention, (ii) a detectable moiety or moieties, and (iii)
reagents and/or solutions to effect and/or detect binding of (i) to an antigen.
30
The present invention further relates to a multivalent composition comprising at least
one polypeptide and comprising at least two antigen binding domains.
Still another aspect of the present invention provides a method for conducting a
35 pharmaceutical business comprising:
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(i) isolating one or more antigen-binding domains that bind to antigens
expressed on the surface of human cells;
(ii) generating a multivalent composition comprising a plurality of said antigen-
binding domains, which multivalent composition kills with an EC 50 of 50nM
5 or less transformed or activated cells where neither cytotoxic entities nor
immunological mechanisms are needed to cause or lead to said killing.;
(iii) conducting therapeutic profiling of the multivalent compositions for efficacy
and toxicity in animals;
(iv) preparing a package insert describing the multivalent composition for
10 treatment of proliferative disorders; and
(v) marketing the multivalent composition for treatment of proliferative
disorders.
The present invention also provides a method for conducting a life science business
15 comprising:
(i) isolating one or more antigen-binding domains that bind to antigens
expressed on the surface of human cells;
(ii) generating a multivalent composition comprising a plurality of said antigen-
binding domains, which multivalent composition kills with an EC 50 of 50nM
20 or less transformed or activated cells where neither cytotoxic entities nor
immunological mechanisms are needed to cause or lead to said killing.;
(iii) licensing, jointly developing or selling, to a third party, the rights for selling
the multivalent compositions.
25 In such embodiments, the the antigen-binding domain can be isolated by a method which
includes
a. isolation of VL and VH domains of human composition from a recombinant
antibody display library by ability to bind to epitopes of HLA DR,
b. generating a library of variants at least one of the CDR1 , CDR2 and CDR3
30 domains of one or both of the VL and VH domains, and
c. isolation of VL and VH domains from the library of variants by ability to
epitopes of HLA DR with a K d of 1 uM or less.
Another business method contemplated by the present invention includes:
35 (i) isolating one or more antigen-binding domains that bind to MHC class II
expressed on the surface of human cells with a Kd of 1 uM or less;
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(ii) generating a composition comprising said antigen-binding domains, which
composition is immunosuppressant with an IC 50 of 100nM or less;
(iii) conducting therapeutic profiling of the multivalent compositions for efficacy
and toxicity in animals;
5 (iv) preparing a package insert describing the use of the composition for
immunosuppression therapy; and
(v) marketing the multivalent composition for use as an immunosuppressant.
The present invention also provides a method for conducting a life science business
10 comprising:
(i) isolating one or more antigen-binding domains that bind to MHC class II
expressed on the surface of human cells with a K d of 1uM or less;
(ii) generating a composition comprising said antigen-binding domains, which
composition is immunosuppressant with an IC 5 o of 100nM or less;
15 (iii) licensing, jointly developing or selling, to a third party, the rights for selling
the compositions.
As used herein, the term "peptide" relates to molecules consisting of one or more
chains of multiple, i. e. two or more, amino acids linked via peptide bonds.
20
The term "protein" refers to peptides where at least part of the peptide has or is able
to acquire a defined three-dimensional arrangement by forming secondary, tertiary, or
quaternary structures within and/or between its peptide chain(s). This definition
comprises proteins such as naturally occurring or at least partially artificial proteins,
25 as well as fragments or domains of whole proteins, as long as these fragments or
domains are able to acquire a defined three-dimensional arrangement as described
above.
The term "polypeptide" is used interchangeably to refer to peptides and/or
30 proteins. Moreover, the terms "polypeptide " and "protein", as the context will admit,
include multi-chain protein complexes, such as immunoglobulin polypeptides having
separate heavy and light chains.
In this context, "polypeptide comprising at least one antibody-based antigen-binding
35 domain" refers to an immunoglobulin (or antibody) or to a fragment thereof. The term
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"fragment", with respect to antibody domains and the like, refers to a fragment of an
immunoglobulin which retains the antigen-binding moiety of an immunoglobulin.
Functional immunoglobulin fragments according to the present invention may be Fv
(Skerra and Pluckthun, 1988), scFv (Bird et al., 1988; Huston et al., 1988), disulfide-
5 linked Fv (Glockshuber et al., 1992; Brinkmann et al., 1993), Fab, F(ab') 2 fragments
or other fragments well-known to the practitioner skilled in the art, which comprise the
variable domains of an immunoglobulin or functional immunoglobulin fragment.
Examples of polypeptides consisting of one chain are single-chain Fv antibody
10 fragments, and examples for polypeptides consisting of multiple chains are Fab
antibody fragments.
The term "antibody" as used herein, unless indicated otherwise, is used broadly to
refer to both antibody molecules and a variety of antibody derived molecules. Such
15 antibody derived molecules comprise at least one variable region (either a heavy
chain of light chain variable region) and include such fragments as described above,
as well as individual antibody light chains, individual antibody heavy chains, chimeric
fusions between antibody chains and other molecules, and the like.
20 The "antigen-binding site" of an immunoglobulin molecule refers to that portion of the
molecule that is necessary for binding specifically to an antigen. An antigen binding
site preferably binds to an antigen with a Kd of 1 p.M or less, and more preferably less
than 100nM, 10nM or even 1nM in certain instances. Binding specifically to an
antigen is intended to include binding to the antigen which significantly higher affinity
25 than binding to any other antigen.
The antigen binding site is formed by amino acid residues of the N-terminal variable
("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains are referred to as "hypervariable
30 regions" which are interposed between more conserved flanking stretches known as
"framework regions," or "FRs". Thus the term "FR" refers to amino acid sequences
which are naturally found between and adjacent to hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable regions of a light
chain and the three hypervariable regions of a heavy chain are disposed relative to
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each other in three dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional surface of a
bound antigen, and the three hypervariable regions of each of the heavy and light
chains are referred to as "complementarity-determining regions," or "CDRs."
5
For the purposes of this application, "valent" refers to the number of antigen binding
sites the subject polypeptide possess. Thus, a bivalent polypeptide refers to a
polypeptide with two binding sites. The term "multivalent polypeptide" encompasses
bivalent, trivalent, tetravalent, etc. forms of the polypeptide.
10
As used herein, a "multivalent composition" means a composition comprising a
polypeptide having at least two of said antigen-binding domains, e.g., a multivalent
polypeptide. Preferably, said at least two antigen-binding domains are in close
proximity so as to mimic the structural arrangement relative to each other of binding
15 sites comprised in a full immunoglobulin molecule. Examples for multivalent
compositions are full immunoglobulin molecules (e.g. IgG, IgA or IgM molecules) or
multivalent fragments thereof (e.g. F(ab') 2 ). Additionally, multivalent compositions of
higher valencies may be formed from two or more multivalent compositions (e.g. two
or more full immunoglobulin molecules), e.g. by cross-linking. Multivalent
20 compositions, however, may be formed as well from two or more monovalent
immunoglobulin fragments, e.g. by self-association as in mini-antibodies, or by cross-
linking.
Accordingly, an "antibody-based antigen-binding domain" refers to polypeptide or
25 polypeptides which form an antigen-binding site retaining at least some of the
structural features of an antibody, such as at least one CDR sequence. In certain
preferred embodiments, antibody-based antigen-binding domain includes sufficient
structure to be considered a variable domain, such as three CDR regions and
interspersed framework regions. Antibody-based antigen-binding domain can be
30 formed single polypeptide chains corresponding to VH or VL sequences, or by
intermolecular or intramolecular association of VH and VL sequences.
The term "recombinant antibody library" describes a variegated library of antigen
binding domains. For instance, the term includes a collection of display packages,
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e.g., biological particles, which each have (a) genetic information for expressing at
least one antigen binding domain on the surface of the particle, and (b) genetic
information for providing the particle with the ability to replicate. For instance, the
package can display a fusion protein including an antigen binding domain. The
5 antigen binding domain portion of the fusion protein is presented by the display
package in a context which permits the antigen binding domain to bind to a target
epitope that is contacted with the display package. The display package will generally
be derived from a system that allows the sampling of very large variegated antibody
libraries. The display package can be, for example, derived from vegetative bacterial
10 cells, bacterial spores, and bacterial viruses.
In an exemplary embodiment of the present invention, the display package is a phage
particle which comprises a peptide fusion coat protein that includes the amino acid
sequence of a test antigen binding domains. Thus, a library of replicable phage
15 vectors, especially phagemids (as defined herein), encoding a library of peptide fusion
coat proteins is generated and used to transform suitable host cells. Phage particles
formed from the chimeric protein can be separated by affinity selection based on the
ability of the antigen binding site associated with a particular phage particle to
specifically bind a target eptipope. In a preferred embodiment, each individual phage
20 particle of the library includes a copy of the corresponding phagemid encoding the
peptide fusion coat protein displayed on the surface of that package. Exemplary
phage for generating the present variegated peptide libraries include M13, f1, fd, If1,
Ike, Xf, Pf1, Pf3, X, T4, 17, P2, P4, ^X-174, MS2 and f2.
25 The term "generating a library of variants of at least one of the CDR1 , CDR2 and
CDR3" refers to a process of generating a library of variant antigen binding sites in
which the members of the library differ by one or more changes in CDR sequences,
e.g., not FR sequences. Such libraries can be generated by random or semi-random
mutagenesis of one or more CDR sequences from a selected antigen binding site.
30
As used herein, an "antibody-based antigen-binding domain of human composition"
preferably means a polypeptide comprising at least an antibody VH domain and an
antibody VL domain, wherein a homology search in a database of protein sequences
comprising immunoglobulin sequences results for both the VH and the VL domain in
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an immunoglobulin domain of human origin as hit with the highest degree of
sequence identity. Such a homology search may be a BLAST search, e.g. by
accessing sequence databases available through the National Center for Biological
Information and performing a "BasicBLAST" search using the "blastp" routine. See
5 also Altschul et al. (1990) J Mol Biol 215:403-410. Preferably, such a composition
does not result in an adverse immune response thereto when administered to a
human recipient. In certain preferred embodiments, the subject antigen-binding
domains of human composition include the framework regions of native human
immunoglobulins, as may be cloned from activated human B cells, though not
1 0 necessarily all of the CDRs of a native human antibody.
As used herein, the term "mini-antibody fragment" means a multivalent antibody
fragment comprising at least two antigen-binding domains multimerized by self-
associating domains fused to each of said domains (Pack, 1994), e.g. dimers
15 comprising two scFv fragments, each fused to a self-associating dimerization domain.
Dimerization domains, which are particularly preferred, include those derived from a
leucine zipper (Pack and Pliickthun, 1992) or helix-turn-helix motif (Pack et al., 1993).
As used herein, "activated cells" means cells of a certain population of interest, which
20 are not resting. Activation might be caused by mitogens (e.g., lipopoysaccharide,
phytohemagglutinine) or cytokines (e.g., interferon gamma). Preferably, said
activation occurs during tumor transformation (e.g., by Epstein-Barr virus, or
"spontaneously"). Preferably, activated cells are characterized by the features of
MHC class II molecules expressed on the cell surface and one or more additional
25 features including increased cell size, cell division, DNA replication, expression of
CD45 or CD11 and production/secretion of immunoglobulin.
As used herein, "non-activated cells" means cells of a population of interest, which
are resting and non-dividing. Said non-activated cells may include resting B cells as
30 purified from healthy human blood. Such cells can, preferably, be characterized by
lack or reduced level of MHC class II molecules expressed on the cell surface and
lack or reduced level of one or more additional features including increased cell size,
cell division, DNA replication, expression of CD45 or CD1 1 and production/secretion
of immunoglobulin.
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As used herein, the term "EC50" means the concentration of multivalent forms of the
subject compositions which produces 50% of its maximum response or effect, such
as cell killing.
5
"At least 5-fold lower EC50" means that the concentration of a multivalent
composition comprising at least one polypeptide of the present invention that is
required to kill 50% of activated cells is at least five times less than the concentration
of the multivalent composition required to kill non-activated cells. Preferably, the
10 concentration required to kill 50% of non-activated cells cannot be achieved with
therapeutically appropriate concentrations of the multivalent composition. Most
preferably, the EC50 value is determined in the test described below in the appended
examples.
15 The term "immunosuppress" refers to the prevention or diminution of the immune
response, as by irradiation or by administration of antimetabolites, antilymphocyte
serum, or specific antibody.
The term "immune response" refers to any response of the immune system, or a cell
20 forming part of the immune system (lymphocytes, granulocytes, macrophages, etc),
to an antigenic stimulus, including, without limitation, antibody production, cell-
mediated immunity, and immunological tolerance.
As used herein, the term "IC50" with respect immunosuppression, refers to the
25 concentration of the subject compositions which produces 50% of its maximum
response or effect, such as inhibition of an immune response, such as may be
manifest by inhibition of IL2 secretion, down-regulation of IL2 expression, or reduced
rate of cell proliferation.
30 The phrase "cytotoxic entities", with reference to a manner of cell killing, refers to
mechanisms which are complement-dependent. Likewise, the phrase "immuological
mechanism" , with reference to a manner of cell killing, refers to macrophage-
dependent and/or neutrophil-dependent killing of cells.
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"Lymphoid cells" when used in reference to a cell line or a cell, means that the cell
line or cell is derived from the lymphoid lineage. "Lymphoid cells" include cells of the
B and the T lymphocyte lineages, and of the macrophage lineage.
5 Cells, which are "non lymphoid cells and express MHC class II", are cells other than
lymphoid cells that express MHC class II molecules, e.g. during a pathological
inflammatory response. For example, said cells may include synovial cells,
endothelial cells, thyroid stromal cells and glial cells, and it may also comprise
genetically altered cells capable of expressing MHC class II molecules.
10
The terms "apoptosis" and "apoptotic activity" refer to the form of cell death in
mammals that is accompanied by one or more characteristic morphological and
biochemical features, including nuclear and condensation of cytoplasm, chromatin
aggregation, loss of plasma membrane microvilli, partition of cytoplasm and nucleus
15 into membrane bound vesicles (apoptotic bodies) which contain ribosomes,
morphologically intact mitochondria and nuclear material, degradation of
chromosomal DNA or loss of mitochondrial function. Apoptosis follows a very
stringent time course and is executed by caspases, a specific group of proteases.
Apoptotic activity can be determined and measured, for instance, by cell viability
20 assays, Annexin V staining or caspase inhibition assays. Apoptosis can be induced
using a cross-linking antibody such as anti-CD95 as described in Example H.
As used herein, the term "first domain of the cc-chain of HLA-DR" means the N-
terminal domain of the alpha-chain of the MHC class II DR molecule.
25
As used herein, the term "first domain of the p-chain of HLA-DR" means the N-
terminal domain of the beta-chain of the MHC class II DR molecule.
The term "innate pre-programmed process" refers to a process that, once it is started,
30 follows an autonomous cascade of mechanisms within a cell, which does not require
any further auxiliary support from the environment of said cell in order to complete the
process.
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As used herein, the term "HuCAL" refers to a fully synthetic human combinatorial
antibody library as described in Knappik et al. (2000).
The term "variable region" as used herein in reference to immunoglobulin molecules
5 has the ordinary meaning given to the term by the person of ordinary skill in the act of
immunology. Both antibody heavy chains and antibody light chains may be divided
into a "variable region" and a "constant region". The point of division between a
variable region and a heavy region may readily be determined by the person of
ordinary skill in the art by reference to standard texts describing antibody structure,
10 e.g., Kabat et al "Sequences of Proteins of Immunological Interest: 5th Edition" U.S.
Department of Health and Human Services, U.S. Government Printing Office (1991).
As used herein, the term "CDR3" refers to the third complementarity-determining
region of the VH and VL domains of antibodies or fragments thereof, wherein the VH
15 CDR3 covers positions 95 to 102 (possible insertions after positions 100 listed as
100a to 100z), and VL CDR3 positions 89 to 96 (possible insertions in VA after
position 95 listed as 95a to 95c) (see Knappik et al., 2000).
As used herein, the term "hybridizes under stringent conditions" is intended to
20 describe conditions for hybridization and washing under which nucleotide sequences
at least 60% homologous to each other typically remain hybridized to each other.
Preferably, the conditions are such that sequences at least 65%, more preferably at
least 70%, and even more preferably at least 75% homologous to each other typically
remain hybridized to each other. Such stringent conditions are known to those skilled
25 in the art and can be found in Current Protocols in Molecular Biology, John Wiley &
Sons, New York. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization conditions is hybridization in 6 x sodium chloride/sodium citrate (SSC) at
about 45°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 50°-65°C.
30 A "protein coding sequence" or a sequence which "encodes" a particular polypeptide
or peptide, is a nucleic acid sequence which is transcribed (in the case of DNA) and
translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed
under the control of appropriate regulatory sequences. The boundaries of the coding
sequence are determined by a start codon at the 5' (amino) terminus and a
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translation stop codon at the 3' (carboxy) terminus. A coding sequence can include,
but is not limited to, cDNA from procaryotic or eukaryotic mRNA, genomic DNA
sequences from procaryotic or eukaryotic DNA, and even synthetic DNA sequences.
A transcription termination sequence will usually be located 3' to the coding
5 sequence.
Likewise, "encodes", unless evident from its context, will be meant to include DNA
sequences which encode a polypeptide, as the term is typically used, as well as DNA
sequences which are transcribed into inhibitory antisense molecules.
10
As used herein, the term "transfection" means the introduction of a heterologous
nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated
gene transfer. "Transient transfection" refers to cases where exogenous DNA does
not integrate into the genome of a transfected cell, e.g., where episomal DNA is
15 transcribed into mRNA and translated into protein. A cell has been "stably
transfected" with a nucleic acid construct when the nucleic acid construct is capable
of being inherited by daughter cells.
"Expression vector" refers to a replicable DNA construct used to express DNA which
20 encodes the desired protein and which includes a transcriptional unit comprising an
assembly of (1) agent(s) having a regulatory role in gene expression, for example,
promoters, operators, or enhancers, operatively linked to (2) a DNA sequence
encoding a desired protein (such as a polypeptide of the present invention) which is
transcribed into mRNA and translated into protein, and (3) appropriate transcription
25 and translation initiation and termination sequences. The choice of promoter and
other regulatory elements generally varies according to the intended host cell. In
general, expression vectors of utility in recombinant DNA techniques are often in the
form of "plasmids" which refer to circular double stranded DNA loops which, in their
vector form are not bound to the chromosome. In the present specification, "plasmid"
30 and "vector" are used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such other forms of
expression vectors which serve equivalent functions and which become known in the
art subsequently hereto.
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In the expression vectors, regulatory elements controlling transcription or translation
can be generally derived from mammalian, microbial, viral or insect genes The ability
to replicate in a host, usually conferred by an origin of replication, and a selection
gene to facilitate recognition of transformants may additionally be incorporated.
5 Vectors derived from viruses, such as retroviruses, adenoviruses, and the like, may
be employed.
"Transcriptional regulatory sequence" is a generic term used throughout the
specification to refer to DNA sequences, such as initiation signals, enhancers, and
10 promoters and the like which induce or control transcription of protein coding
sequences with which they are operably linked. It will be understood that a
recombinant gene can be under the control of transcriptional regulatory sequences
which are the same or which are different from those sequences which control
transcription of the naturally-occurring form of the gene, if any.
15
"Operably linked" when describing the relationship between two DNA regions simply
means that they are functionally related to each other. For example, a promoter or
other transcriptional regulatory sequence is operably linked to a coding sequence if it
controls the transcription of the coding sequence.
20
As used herein, the term "fusion protein" is art recognized and refer to a chimeric
protein which is at least initially expressed as single chain protein comprised of amino
acid sequences derived from two or more different proteins, e.g., the fusion protein is
a gene product of a fusion gene.
25
As used herein, "proliferating" and "proliferation" refer to cells undergoing mitosis.
The "growth rate" of a cell refers to the rate of proliferation of the cell and the state of
differentiation of the cell.
30
The term "cell-proliferative disorder" denotes malignant as well as nonmalignant
populations of transformed cells which morphologically often appear to differ from the
surrounding tissue.
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As used herein, "transformed cells" refers to cells which have spontaneously
converted to a state of unrestrained growth, i.e., they have acquired the ability to grow
through an indefinite number of divisions in culture. Transformed cells may be
characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with
5 respect to their loss of growth control.
As used herein, "immortalized cells" refers to cells which have been altered via
chemical and/or recombinant means such that the cells have the ability to grow
through an indefinite number of divisions in culture.
10
As used herein the term "animal" refers to mammals, preferably mammals such as
humans. Likewise, a "patient" or "subject" to be treated by the method of the
invention can mean either a human or non-human animal.
15 According to the methods of the invention, the peptide may be administered in a
pharmaceutically acceptable composition. In general, pharmaceutically-acceptable
carriers for monoclonal antibodies, antibody fragments, and peptides are well-known
to those of ordinary skill in the art. As used herein, the term "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion media, coatings,
20 antibacterial and antifungal agents, isotonic and absorption delaying agents and the
like. In preferred embodiments, the subject carrier medium which does not interfere
with the effectiveness of the biological activity of the active ingredients and which is
not excessively toxic to the hosts of the concentrations of which it is administered.
The administration(s) may take place by any suitable technique, including
25 subcutaneous and parenteral administration, preferably parenteral. Examples of
parenteral administration include intravenous, intraarterial, intramuscular, and
intraperitoneal, with intravenous being preferred.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions
30 or dispersions and sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be stable under the conditions
of manufacture and storage and must be preserved against the contaminating action
of microorganisms, such as bacteria and fungi. The carrier can be a solvent or
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dispersion medium containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for
example, by the use of a coating, such as lecithin, by the maintenance of the required
5 particle size in the case of dispersion and by the use of surfactants. The prevention of
the action of microorganisms can be brought about by various antibacterial an
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it will be preferable to include isotonic agents,
for example, sugars or sodium chloride. Prolonged absorption of the injectable
1 0 compositions can be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds, e.g.,
the subject polypeptides, in the required amount in the appropriate solvent with
15 various of the other ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the various
sterilized active ingredients into a sterile vehicle which contains the basic dispersion
medium and the required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable solutions, the preferred
20 methods of preparation are vacuum-drying and freeze-drying techniques which yield a
powder of the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
For oral administration the polypeptides of the present invention may be incorporated
25 with excrpients and used in the form of non-ingestible mouthwashes and dentifrices. A
mouthwash may be prepared incorporating the active ingredient in the required
amount in an appropriate solvent, such as a sodium borate solution (Dobell's
Solution). The active ingredient may also be dispersed in dentifrices, including: gels,
pastes, powders and slurries. The active ingredient may be added in a therapeutically
30 effective amount to a paste dentifrice that may include water, binders, abrasives,
flavoring agents, foaming agents, and humectants.
The compositions of the present invention may be formulated in a neutral or salt form.
Pharmaceutically-acceptable salts include the acid addition salts (formed with the free
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amino groups of the protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium, potassium, ammonium,
5 calcium, or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
For parenteral administration in an aqueous solution, for example, the solution should
be suitably buffered if necessary and the liquid diluent first rendered isotonic with
10 sufficient saline or glucose. These particular aqueous solutions are especially suitable
for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In
this connection, sterile aqueous media which can be employed will be known to those
of skill in the art in light of the present disclosure. For example, one dosage could be
dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of
1 5 hypodermoclysis fluid or injected at the proposed site of infusion, (see for example,
"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-
1580). Some variation in dosage will necessarily occur depending on the condition of
the subject being treated. The person responsible for administration will, in any event,
determine the appropriate dose for the individual subject. Moreover, for human
20 administration, preparations should meet sterility, pyrogenicity, general safety and
purity standards as required by FDA Office of Biologies standards.
Upon formulation, solutions can be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically effective: The
25 formulations are easily administered in a variety of dosage forms such as injectable
solutions, drug release capsules and the like.
As used herein, the term "prophylactic or therapeutic" treatment refers to
administration to the host of the medical condition. If it is administered prior to
30 exposure to the condition, the treatment is prophylactic (i.e., it protects the host
against tumor formation), whereas if administered after initiation of the disease, the
treatment is therapeutic (i.e., it combats the existing tumor).
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A multivalent composition of at least one polypeptide according to the invention is
capable of causing cell death of activated cells, preferably lymphoid tumor cells
without requiring any further additional measures such as chemotherapy and with
limited immunogenic side effects on the treated patient. Further, the multivalent
5 composition comprising a polypeptide according to the invention has the capability of
binding to at least one epitope on the target antigen, however, several epitope binding
sites might be combined in one molecule. Preferably, the multivalent composition
comprising a polypeptide according to the invention shows at least 5-fold, or more
preferably 10-fold higher killing activity against activated cells compared to non-
10 activated cells. This higher activity on activated cells can be expressed as the at least
5-fold lower EC50 value on activated versus non-activated cells or as the higher
percentage of killing of activated cells versus non-activated cells when using the
same concentration of protein. Under the latter alternative, the multivalent
composition comprising a polypeptide according to the invention at a given
15 polypeptide concentration kills at least 50%, preferably at least 80%, of activated
cells, whereas the same concentration of a multivalent composition comprising a
polypeptide according to the invention under the same incubation conditions kills less
than 15%, preferably less than 10% of the non-activated cells. The assay conditions
for determining the EC50 value and the percentage killing activity are described
20 below.
Brief Description of the Drawings
Figure 1
25 a. Specificity of the anti-HLA-DR antibody fragments: Binding of MS-GPC-8-27-7,
MS-GPC-8-27-10, MS-GPC-8-6-13, MS-GPC-8-27-41 , MS-GPC-8-6-47, MS-GPC-8-
10-57, MS-GPC-8-6-27, MS-GPC-8 and MS-GPC-8-6 to HLA-DR protein, negative
control proteins (BSA, testosterone-BSA, lysozyme and human apotransferrin), and
an empty microtiter plate well (plastic). Specificity was assessed using standard
30 ELISA procedures.
b. Specificity of the anti-HLA-DR antibody fragments MS-GPC-1, 6, 8 & 10 isolated
from the HuCAL library to HLA-DR protein, a mouse-human chimeric HLA protein
and negative control proteins (lysozyme, transferrin, BSA and human B~globulin).
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Specificity was assessed using standard ELISA procedures. A non-related antibody
fragment (irr. scFv) was used as control.
Figure 2
5 Reactivity of the anti-HLA-DR antibody fragments (MS-GPC-1 , 6, 8 and 10) and IgG
forms of MS-GPC-8, MS-GPC-8-10-57, MS-GPC-8-27-41 & MS-GPC-8-6-1 7 to
various cell lines expressing MHC class II molecules. "+" represents strong reactivity
as detected using standard immunofluorescence procedure. "+/-" represents weak
reactivity and "-" represents no detected reactivity between an anti-HLA-DR antibody
10 fragment or IgG and a particular cell line.
Figure 3
Viability of tumor cells in the presence of monovalent and cross-linked anti-HLA-DR
antibody fragments as assessed by trypan blue staining. Viability of GRANTA-519
15 cells was assessed after 4 h incubation with anti-HLA-DR antibody fragments (MS-
GPC-1, 6, 8 and 10) with and without anti-FLAG M2 mAb as cross-linking agent.
Figure 4
Scatter plots and fitted logistic curves of data from Table 5 showing improved killing
20 efficiency of 50 nM solutions of the IgG form of the human antibody fragments of the
invention treated compared to treatment with 200 nM solutions of murine antibodies.
Open circles represent data for cell lines treated with the murine antibodies L243 and
8D1 and closed circles for human antibodies MS-GPC-8, MS-GPC-8-27-41, MS-
GPC-8-10-57 and MS-GPC-8-6-1 3. Fitted logistic curves for human (solid) and
25 mouse (dashed) mAb cell killing data show the overall superiority of the treatment
with human mAbs at 50 nM compared to the mouse mAbs despite treatment at a final
concentration of 200 nM.
30 Figure 5
Killing of activated versus non-activated cells. MHH-PREB-1 cells are activated with
Lipopolysaccharide, Interferon-gamma and phyto-hemagglutin, and subsequently
incubated for 4 h with 0.07 to 3300 nM of the IgG forms of the anti-HLA-DR antibody
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fragments MS-GPC-8-10-57 and MS-GPC-8-27-41 . No loss of viability in the control
non-activated MHH-PREB-1 cells is seen.
Figure 6
5 Killing efficiency of control (no antibody, unreactive murine IgG; light grey), and
human (MS-GPC-8, MS-GPC-8-1 0-57 & MS-GPC-8-27-41 ; dark grey) IgG forms of
anti-HLA-DR antibody fragments against CLL cells isolated from patients. Left panel,
box-plot display of viability data from 10 patient resting cell cultures against antibodies
after incubation for four (h4) and twenty four hours (h24). Right panel box-plot display
10 of viability data from 6 patient activated cell cultures against antibodies after
incubation for four (h4) and twenty four hours (h24).
Figure 7
15 Concentration dependent cell viability for certain anti-HLA-DR antibody fragments of
the invention. Vertical lines indicate the EC50 value estimated by logistic non-linear
regression on replica data obtained for each of the antibody fragments, a) Killing
curves of cross-linked bivalent anti-HLA-DR antibody F(ab) fragment dimers MS-
GPC-10 (circles and solid line), MS-GPC-8 (triangles and dashed line) and MS-GPC-
20 1 (crosses and dotted line), b) Killing curves of cross-linked bivalent anti-HLA-DR
antibody (Fab) fragment dimers MS-GPC-8-1 7 (circles and solid line), and murine
IgGs 8D1 (triangles and dashed line) and L243 (crosses and dotted line), c) Killing
curves of cross-linked bivalent anti-HLA-DR antibody (Fab) fragment dimers GPC-8-
6-2 (crostriangles and dashed line), and murine IgGs 8D1 (circles and solid line) and
25 L243 (crosses and dotted line), d) Killing curves of IgG forms of human anti-HLA-DR
antibody fragments MS-GPC-8-10-57 (crosses and dotted line), MS-GPC-8-27-41
(exes and dash-dot line), and murine IgGs 8D1 (circles and solid line) and L243
(triangles and dashed line). All concentrations are given in nM of the bivalent agent
(IgG or cross-linked (Fab) dimer).
30
Figure 8
a. Incubation of Priess cells with the anti-HLA-DR antibody fragment MS-GPC-8,
cross-linked using the anti-FLAG M2 mAb, shows more rapid killing than a culture of
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Priess cells induced into apoptosis using anti-CD95 mAb. An Annexin V/PI staining
technique identifies necrotic cells by Annexin V positive and PI positive staining.
b. Incubation of Priess cells with the anti-HLA-DR antibody fragment MS-GPC-8,
5 cross-linked using the anti-FLAG M2 mAb, shows little evidence of an apoptotic
mechanism compared to an apoptotic culture of Priess cells induced using anti-CD95
mAb. An Annexin V/PI staining technique identifies apoptotic cells by Annexin V
positive and PI negative staining.
1 0 Figure 9
a. Immunosuppressive properties of the IgG forms of the anti-HLA-DR antibody
fragments MS-GPC-8-10-57, MS-GPC-8-27-41 & MS-GPC-8-6-13 using an assay to
determine inhibition of IL-2 secretion from T-hybridoma cells.
b. Immunosuppressive properties of the monovalent Fab forms of the anti-HLA-DR
15 antibody fragments MS-GPC-8-27-41 & MS-GPC-8-6-1 9 using an assay to determine
inhibition of IL-2 secretion from T-hybridoma cells
Figure 10
Immunosuppressive properties of the IgG forms of the anti-HLA-DR antibody
20 fragments MS-GPC-8-10-57 and MS-GPC-8-27-41 in an assay to determine inhibition
of T cell proliferation.
Figure 1 1
Vector map and sequence of scFv phage display vector pMORPH13_scFv.
25 The vector pMORPH13_scFv is a phagemid vector comprising a gene encoding a
fusion between the C-terminal domain of the gene III protein of filamentous phage
and a HuCAL scFv. In Figure 11, a vector comprising a model scFv gene
(combination of VH1A and VX3 (Knappik et al., 2000) is shown.
The original HuCAL master genes (Knappik et al. (2000): see Fig. 3 therein) have
30 been constructed with their authentic N-termini: VH1A, VH1B, VH2, VH4 and VH6
with Q (=CAG) as the first amino acid. VH3 and VH5 with E (=GAA) as the first amino
acid. Vector pMORPH13_scFv comprises the short FLAG peptide sequence (DYKD)
fused to the VH chain, and thus all HuCAL VH chains in, and directly derived from,
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this vector have E (=GAA) at the first position (e.g. in pMx7_FS vector, see Figure
12).
Figure 12
5 Vector map and sequence of scFv expression vector pMx7_FS_5D2.
The expression vector pMx7_FS_5D2 leads to the expression of HuCAL scFv
fragments (in Figure 12, the vector comprises a gene encoding a "dummy" antibody
fragment called "5D2") when VH-CH1 is fused to a combination of a FLAG tag (Hopp
et al., 1988; Knappik and Pluckthun, 1994) and a STREP tag II (WSHPQFEK) (IBA
10 GmbH, Gottingen, Germany; see: Schmidt and Skerra, 1993; Schmidt and Skerra,
1994; Schmidt et a!., 1996; Voss and Skerra, 1997).
Figure 13
Vector map and sequence of Fab expression vector pMx9_Fab_GPC8.
15 The expression vector pMx9_Fab_GPC8 leads to the expression of HuCAL Fab
fragments (in Figure 13, the vector comprises the Fab fragment MS-GPC8) when VH-
CH1 is fused to a combination of a FLAG tag (Hopp et al., 1988; Knappik and
Pluckthun, 1994) and a STREP tag II (WSHPQFEK) (IBA GmbH, Gottingen,
Germany; see: Schmidt and Skerra, 1993; Schmidt and Skerra, 1994; Schmidt et al.,
20 1 996; Voss and Skerra, 1 997).
In pMx9_Fab vectors, the HuCAL Fab fragments cloned from the scFv fragments
(see figure caption of Figure 11) do not have the short FLAG peptide sequence
(DYKD) fused to the VH chain, and ail HuCAL VH chains in, and directly derived from,
that vector have Q (=CAG) at the first position
25
Figure 14
Vector map and sequence of Fab phage display vector pMORPH18 Fab_GPC8.
The derivatives of vector pMORPH18 are phagemid vectors comprising a gene
encoding a fusion between the C-terminal domain of the gene III protein of
30 filamentous phage and the VH-CH1 chain of a HuCAL antibody. Additionally, the
vector comprises the separately encoded VL-CL chain. In Figure 14, a vector
comprising the Fab fragment MS-GPC-8 is shown.
In pMORPH18_Fab vectors, the HuCAL Fab fragments cloned from the scFv
fragments (see figure caption of Figure 11) do not have the short FLAG peptide
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sequence (DYKD) fused to the VH chain, and all HuCAL VH chains in, and directly
derived from, that vector have Q (=CAG) at the first position.
Figure 15
5 Amino acid sequences of VH and VL domains of MS-GPC-1 , MS-GPC-6, MS-GPC-8,
MS-GPC-10, MS-GPC-8-6, MS-GPC-8-10, MS-GPC-8-1 7, MS-GPC-8-27, MS-GPC-
8-6-13, MS-GPC-8-1 0-57, and MS-GPC-8-27-41 .
The sequences in Figure 15 show amino acid 1 of VH as constructed in the original
HuCAL master genes (Knappik et al. (2000): see Fig. 3 therein). In scFv constructs,
10 as described in this application, amino acid 1 of VH is always E (see figure caption of
Figure 11), in Fab constructs as described in this application, amino acid 1 of VH is
always Q (see figure caption of Figure 13)
Detailed Description of the invention
1 5 The following examples illustrate the invention.
Examples
All buffers, solutions or procedures without explicit reference can be found in standard
textbooks, for example Current Protocols of Immunology (1997 and 1999) or
20 Sambrook et al., 1989. Where not given otherwise, all materials were purchased from
Sigma, Deisenhofen, DE, or Merck, Darmstadt, DE, or sources are given in the
literature cited. Hybridoma cell lines LB3.1 and L243 were obtained from LGC
Reference Materials, Middlesex, UK; data on antibody 8D1 were generously supplied
by Dr. Matyas Sandor, University of Michigan, Madison, Wl, USA.
25
1. Preparation of a human antigen
To demonstrate that we could identify cytotoxic antigen-binding domains of human
composition, we first prepared a purified form of a human antigen, the human MHC
class II DR protein (DRA*0101/DRB1*0401) from PRIESS cells (Gorga et al., 1984;
30 Gorga et al., 1986; Gorga et al., 1987; Stern et al., 1992) as follows.
First, PRIESS cells (ECACC, Salisbury UK) were cultured in RPMI and 10% fetal calf
serum (FCS) using standard conditions, and 10 10 cells were lysed in 200 ml
phosphate buffered saline (PBS) (pH 7.5) containing 1% NP-40 (BDH, Poole, UK), 25
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mM iodoacetamide, 1 mM phenylmethylsulfonylfluoride (PMSF) and 10 mg/l each of
the protease inhibitors chymostatin, antipain, pepstatin A, soybean trypsin inhibitor
and leupeptin. The lysate was centrifuged at 10.000 g (30 minutes, 4°C) and the
resulting supernatant was supplemented with 40 ml of an aqueous solution
5 containing 5% sodium deoxycholate, 5 mM iodoacetamide and 10 mg/l each of the
above protease inhibitors and centrifuged at 100.000 g for two hours (4°C). To
remove material that bound non-specifically and endogenous antibodies, the resulting
supernatant was made 0.2 mM with PMSF and passed overnight (4°C) through a
rabbit serum affigel-10 column (5 ml; for preparation, rabbit serum (Charles River,
10 Wilmington, MA, USA) was incubated with Affigel 10 (BioRad, Munich, DE) at a
volume ratio of 3:1 and washed following manufacturer's directions) followed by a
Protein G Sepharose Fast Flow column (2 ml; Pharmacia) using a flow rate of 0.2
ml/min.
Second, the pre-treated lysate was batch incubated with 5 ml Protein G Sepharose
Fast Flow beads coupled to the murine anti-HLA-DR antibody LB3.1 (obtained by
Protein G-Sepharose FF (Pharmacia) affinity chromatography of a supernatant of
hybridoma cell line LB3.1) (Stern et al„, 1993) overnight at 4°C using gentle mixing,
and then transferred into a small column which was then washed extensively with
three solutions: (1) 100 ml of a solution consisting of 50 mM Tris/HCI (pH 8.0), 150
mM NaCI, 0.5% NP-40, 0.5% sodium deoxycholate, 10% glycerol and 0.03% sodium
azide at a flow rate of 0.6 ml/min). (2) 25 ml of a solution consisting of 50 mM
Tris/HCI (pH 9.0), 0.5 M NaCI, 0.5% NP-40, 0.5% sodium deoxycholate, 10%
glycerol and 0.03% sodium azide at a flow rate of 0.9 ml/min; (3) 25 ml of a solution
consisting of 2 mM Tris/HCI (pH 8.0), 1% octyl-R-D-glucopyranoside, 10% glycerol
and 0.03% sodium azide at a flow rate of 0.9 ml/min.
Third, MHC class II DR protein (DRA*0101/DRB1*0401) was eluted using 15 ml of a
solution consisting of 50 mM diethylamine/HCI (pH 11.5), 150 mM NaCI, 1 mM EDTA,
30 1 mM EGTA, 1% octyl-G-D-glucopyranoside (Alexis Corp., Lausen, CH), 10%
glycerol, 10 mM iodoacetamide and 0.03% sodium azide at a flow rate of 0.4 ml/min.
800 pi fractions were immediately neutralised with 100 pi 1M Tris/HCI (pH 6.8), 150
mM NaCI and 1% octyl-ft-D-glucopyranoside. The incubation of the lysate with LB3.1-
Protein G Sepharose Fast Flow beads was repeated until the lysate was exhausted of
40
15
20
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MHC protein. Pure eluted fractions of the MHC class II DR protein (as analyzed by
SDS-PAGE) were pooled and concentrated to 1.0-1.3 g/l using Vivaspin
concentrators (Greiner, Solingen, DE) with a 30 kDa molecular weight cut-off.
Approximately 1 mg of the MHC class II DR preparation was re-buffered with PBS
5 containing 1% octyl-B-D-glucopyranoside using the same Vivaspin concentrator to
enable direct coupling of the protein to BIAcore CM5 chips.
2. Screening ofHuCAL
2.1. Introduction
10 We identified certain antigen binding antibody fragments of human composition (MS-
GPC-1, MS-GP-6, MS-GPC-8 and MS-GPC-10) against the human antigen
(DRA*01 01 /DRB 1*0401) from a human antibody library based on a novel concept
that has been recently developed (Knappik et al., 2000). A consensus framework
resulting in a total of 49 different frameworks here represents each of the VH- and
15 VL-subfamilies frequently used in human immune responses. These master genes
were designed to take into account and eliminate unfavorable residues promoting
protein aggregation as well as to create unique restriction sites leading to modular
composition of the genes. In HuCAL-scFv, both the VH- and VL-CDR3 encoding
regions of the 49 master genes were randomized.
20
2.2. Phagemid rescue, phage amplification and purification
The HuCAL-scFv (Knappik et al., 2000) library, cloned into a phagem id-based phage
display vector pMORPH13_scFv (see Figure 11), in E.coli TG-1 was amplified in 2 x
TY medium containing 34 ug/ml chloramphenicol and 1 % glucose (2 x TY-CG). After
25 helper phage infection (VCSM13) at 37°C at an OD 6 oo of about 0.5, centrifugation and
resuspension in 2 x TY / 34 ug/ml chloramphenicol / 50 ug/ml kanamycin / 0.1 mM
IPTG, cells were grown overnight at 30°C. Phage were PEG-precipitated from the
supernatant (Ausubel et al., 1998), resuspended in PBS/20% glycerol and stored at-
80°C. Phage amplification between two panning rounds was conducted as follows:
30 mid-log phase TG1 -cells were infected with eluted phage and plated onto LB-agar
supplemented with 1 % of glucose and 34 ug/ml of chloramphenicol. After overnight
incubation at 30°C colonies were scraped off, adjusted to an OD 60 o of 0.5 and helper
phage added as described above.
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2.3. Manual solid phase panning
Wells of MaxiSorp™ microtiterplates (Nunc, Roskilde, DK) were coated with MHC-
class II DRA*0101/DRB1*0401 (prepared as above) dissolved in PBS (2 ug/well).
After blocking with 5% non-fat dried milk in PBS, 1—5 x 10 12 HuCAL-scFv phage
5 purified as above were added for 1h at 20°C. After several washing steps, bound
phages were eluted by pH-elution with 100 mM triethylamine and subsequent
neutralization with 1 M TRiS-CI pH 7.0. Three rounds of panning were performed with
phage amplification conducted between each round as described above.
10 2.4. Mixed solid phase/whole cell panning
Three rounds of panning and phage amplification were performed as described in 2.3.
and 2.2. with the exception that in the second round between 1 x 10 7 and 5 x 10 7
PRIESS cells in 1 ml PBS/10% FCS were used in 10 ml Falcon tubes for whole cell
panning. After incubation for 1h at 20°C with the phage preparation, the cell
15 suspension was centrifuged (2000 rpm for 3 min) to remove non-binding phage, the
cells were washed three times with 10 ml PBS, each time followed by centrifugation
as described. Phage that specifically bound to the cells were eluted off by pH-elution
using 1 00 mM HCI. Alternatively, binding phage could be amplified by directly adding
E.coli to the suspension after triethlyamine treatment (100 mM) and subsequent
20 neutralization.
2.5 Identification of HLA-DR binding scFv fragments
Clones obtained after three rounds of solid phase panning (2.3) or mixed solid
phase/whole cell panning (2.4) were screened by FACS analysis on PRIESS cells for
25 binding to HLA-DR on the cell surface. For expression, the scFv fragments were
cloned via Xba l /Eco RI into pMx7_FS as expression vector (see Figure 12).
Expression conditions are shown below in example 3.2
Aliquots of 10 6 Priess cells were transferred at 4°C into wells of a 96-well
30 microtiterplate. ScFv in blocking buffer (PBS/5% FCS) were added for 60 min and
detected using an anti-FLAG M2 antibody (Kodak) (1:5000 dilution) followed by a
polyclonal goat anti-mouse IgG antibody-R-Phycoerythrin-conjugate (Jackson
ImmunoResearch, West Grove, PA, USA, Cat. No. 115-116-146, F(ab')2 fragment)
(1:200 dilution). Cells were fixed in 4% paraformaldehyde for storage at 4°C. 10 4
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events were collected for each assay on the FACS-Calibur (BD Immunocytometry
Systems, San Jose, CA, USA).
Only fifteen out of over 500 putative binders were identified which specifically bound
5 to Priess cells. These clones were further analyzed for their killing activity as
described below. Table 1 contains the sequence characteristics of clones MS-GPC-1 ,
MS-GPC-6, MS-GPC-8 and MS-GPC-1 0 identified thereby. The VH and VL families
and the CDR3s listed refer to the HuCAL consensus-based antibody genes as
described (Knappik et al., 2000); the sequences of the VH and VL CDRs are shown in
10 Table 1, and the full sequences of the VH and VL domains are shorn in Figure 15.
3. Generation of Fab-fragments
3.1 . Conversion of scFv to Fab
The Fab-fragment antigen binding polypeptides MS-GPC-1 -Fab, MS-GP-6-Fab, MS-
15 GPC-8-Fab and MS-GPC-1 0-Fab were generated from their corresponding scFv
fragments as follows. Both heavy and light chain variable domains of scFv fragments
were cloned into pMx9_Fab (Figure 13), the heavy chain variable domains as Mfe l /
Styl-fragments, the variable domains of the kappa light chains as Eco RV/ BsiWI-
fragments. The lambda chains were first amplified from the corresponding
20 pMORPH13_scFv vector as template with PCR-primers CRT5 (5' primer) and CRT6
(3' primer), wherein CRT6 introduces a unique Dra lll restriction endonuclease site.
CRT5: 5' GTGGTGGTTCCGy4L47"C 3'
25 CRT6: 5' AG CGTC AC/4 CTCGGTG CG G CTTTCG G CTG G CCAAG AACG G GTTA 3'
The PCR product is cut with Eco RV / Dralll and cloned into pMx9_Fab (see Figure
13). The Fab light chains could be detected with a polyclonal goat anti-human IgG
antibody-R-Phycoerythrin-conjugate (Jackson ImmunoResearch, West Grove, PA,
30 USA, Cat. No. 1 09-1 1 6-088, F(ab') 2 fragment) (1 :200 dilution).
3.2. Expression and purification of HuCAL-antibody fragments in E.coli
Expression in E.co li cells (JM83) of scFv and Fab fragments from pMx7_FS or
pMx9_Fab, respectively, were carried out in one litre of 2 x TY-medium supplemented
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with 34 |jg/ml chloramphenicol. After induction with 0.5 mM IPTG (scFv) or 0.1 mM
IPTG (Fab), cells were grown at 22°C for 12 hours. Cell pellets were lysed in a French
Press (Thermo Spectronic, Rochester, NY, USA) in 20 mM sodium phosphate, 0.5 M
NaCI, and 10 mM imidazole (pH 7.4). Cell debris was removed by centrifugation and
5 the clear supernatant filtered through 0.2 urn pores before subjecting it to STREP tag
purification using a Streptactin matrix and purification conditions according to the
supplier (IBA GmbH, Gottingen, Germany). Purification by size exclusion
chromatography (SEC) was performed as described by Rheinnecker et al. (1996).
The apparent molecular weights were determined by SEC with calibration standards
10 and confirmed in some instances by coupled liquid chromatography-mass
spectrometry (TopLab GmbH, Martinsried, Germany).
4. Optimization of antibody fragments
In order to optimize certain biological characteristics of the HLA-DR binding antibody
15 fragments, one of the Fab fragments, MS-GPC-8-Fab, was used to construct a library
of Fab antibody fragments by replacing the parental VL A1 chain by the pool of all
lambda chains A 1-3 randomized in CDR3 from the HuCAL library (Knappik et al.,
2000).
20 The Fab fragment MS-GPC-8-Fab (see 3.1) was cloned via Xbal/EcoRI from
pMx9_Fab_GPC-8 into pMORPH18_Fab, a phagemid-based vector for phage display
of Fab fragments, to generate pMORPH18_Fab_GPC-8 (see Figure 14). A lambda
chain pool comprising a unique Dra lll restriction endonuclease site (Knappik et al.,
2000) was cloned into pMORPH18_Fab_GPC-8 cut with Nsil and Dralll (see vector
25 map of pMORPH18J=ab_GPC-8 in Figure 14).
The resulting Fab optimization library was screened by two rounds of panning against
MHC-class II DRA*0101/DRB1*0401 (prepared as above) as described in 2.3 with the
exception that in the second round the antigen concentration for coating was
30 decreased to 12 ng/well. FACS identified optimized clones as described above in 2.5.
Seven of these clones, MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10,
MS-GPC-8-17, MS-GPC-8-1 8 and MS-GPC-8-27, were further characterized and
showed cell killing activity as found for the starting fragment MS-GPC-8. Table 1
contains the sequence characteristics of MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9,
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MS-GPC-8-10, MS-GPC-8-17, MS-GPC-8-18 and MS-GPC-8-27. The VH and VL
families and the CDR3s listed refer to the HuCAL consensus-based antibody genes
as described (Knappik et al., 2000). The full sequences of the VH and VL domains of
MS-GPC-8-6, MS-GPC-8-10, MS-GPC-8-17 and MS-GPC-8-27are shown in Figure
5 15.
The optimized Fab forms of the anti-HLA-DR antibody fragments MS-GPC-8-6 and
MS-GPC-8-17 showed improved characteristics over the starting MS-GPC-8. For
example, the EC50 of the optimized antibodies was 15-20 and 5-20 nM (compared to
1 0 20-40 nM for MS-GPC-8, where the concentration is given as the concentration of the
bivalent cross-linked Fab dimer), and the maximum capacity to kill MHH-Call 4 cells
determined as 76 and 78% for MS-GPC-8-6 and MS-GPC-8-17 (compared to 65% for
MS-GPC-8) respectively.
15 For further optimization, the VL CDR1 regions of a set of anti-HLA-DR antibody
fragments derived from MS-GPC-8 (including MS-GPC-8-10 and MS-GPC-8-27) were
optimized by cassette mutagenesis using trinucleotide-directed mutagenesis
(Vimekas et al., 1994). In brief, a VI1 CDR1 library cassette was synthesized
containing six randomized positions (total variability: 7.43 x 10 6 ), and was cloned into
20 a VI1 framework. The CDR1 library was digested with Eco RV and Bbs l. and the
fragment comprising the CDR1 library ligated into the lambda light chains of the MS-
GPC-8-derived Fab antibody fragments in pMORPH18_Fab (as described above),
digested with Eco RV and Bbs l. The resulting library was screened as described
above. Ten clones were identified as above by binding specifically to HLA DR (MS-
25 GPC-8-6-2, MS-GPC-8-6-19, MS-GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-13,
MS-GPC-8-6-47, MS-GPC-8- 10-57, MS-GPC-8-27-7, MS-GPC-8-27-10 & MS-GPC-
8-27-41) and showed cell killing activity as found for the starting fragments MS-GPC-
8, MS-GPC-8-10 and MS-GPC-8-27. Table 1 contains the sequence characteristics of
MS-GPC-8-6-2, MS-GPC-8-6-19, MS-GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-
30 13, MS-GPC-8-6-47, MS-GPC-8-10-57, MS-GPC-8-27-7, MS-GPC-8-27-10 & MS-
GPC-8-27-41. The VH and VL families and the CDR3s listed refer to the HuCAL
consensus-based antibody genes as described (Knappik et al., 2000), the full
sequences of the VH and VL domains of MS-GPC-8-6-13, MS-GPC-8-10-57 & MS-
GPC-8-27-41 are shown in Figure 15.
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From these 10 clones, four Fab fragments were chosen (MS-GPC-8-6-2, MS-GPC-8-
6-13, MS-GPC-8-10-57 and MS-GPC-8-27-41) as demonstrating significantly
improved EC50 of cell killing as described in example 10. Table 1 shows the
5 sequences of clones optimised at the CDR1 region.
Optimisation procedures not only increased the biological efficacy of anti-HLA DR
antibody fragments generated by the optimisation process, but a physical
characteristic - affinity of the antibody fragment to HLA DR protein - was also
10 substantially improved. For example, the affinity of Fab forms of MS-GPC-8 and its
optimised descendents was measured using a surface plasmon resonance
instrument (Biacore, Upsala Sweden) according to example 7. The affinity of the MS-
GPC-8 parental Fab was improved over 100 fold from 346 nM to ~ 60 nM after
VLCDR3 optimisation and further improved to single digit nanomolar affinity (range 3
15 - 9 nM) after VLCDR3+1 optimisation (Table 2).
5. Generation of IgG
5.1 Construction of HuCAL-immunoglobulin expression vectors
20 Heavy chains were cloned as follows. The multiple cloning site of pcDNA3.1 +
(Invitrogen) was removed (Nhel / Apa l), and a stuffer compatible with the restriction
sites used for HuCAL-design was inserted for the ligation of the leader sequences
(Nhe l / EcoRI), VH-domains ( Eco RI / Blp l) and the immunoglobulin constant regions
( Blp l / Apa l). The leader sequence (EMBL M83133) was equipped with a Kozak
25 sequence (Kozak, 1987). The constant regions of human lgG1 (PIR J00228), lgG4
(EMBL K01316) and serum lgA1 (EMBL J00220) were dissected into overlapping
oligonucleotides with lengths of about 70 bases. Silent mutations were introduced to
remove restriction sites non-compatible with the HuCAL-design. The oligonucleotides
were spliced by overlap extension-PCR.
30
Light chains were cloned as follows. The multiple cloning site of pcDNA3.1/Zeo+
(Invitrogen) was replaced by two different stuffers. The K-stuffer provided restriction
sites for insertion of a K-leader (Nhe l / Eco RV), HuCAL-scFv VK-domains (EcoRV /
BsiWI) and the K-chain constant region (Bsi WI / Apa l). The corresponding restriction
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sites in the A-stuffer were Nhe l / Eco RV (A-leader), Eco RV / Hpa l (VA- domains) and
Hpa l / Apa l (A-chain constant region). The K-leader (EMBL Z00022) as well as the A-
leader (EMBL L27692) were both equipped with Kozak sequences. The constant
regions of the human k- (EMBL J00241) and A -chain (EMBL M18645) were
5 assembled by overlap extensfon-PCR as described above.
5.2 Generation of IgG-expressing CHO-cells
All cells were maintained at 37°C in a humidified atmosphere with 5% C02 in media
recommended by the supplier. CHO-K1 (CRL-961 8) were from ATCC and were co-
10 transfected with an equimolar mixture of IgG heavy and light chain expression
vectors. Double-resistant transfectants were selected with 600 ug/ml G418 and 300
ug/ml Zeocin (Invitrogen) followed by limiting dilution. The supernatant of single
clones was assessed for IgG expression by capture-ELISA. Positive clones were
expanded in RPMI-1640 medium supplemented with 10% ultra-low IgG-FCS (Life
15 Technologies). After adjusting the pH of the supernatant to 8.0 and sterile filtration,
the solution was subjected to standard protein A column chromatography (Poros 20A,
PE Biosystems).
The IgG forms of anti-HLA-DR antigen binding domains show improved
20 characteristics over the antibody fragments. These improved characteristics include
affinity (Example 7) and killing efficiency (Examples 9, 10 and 14).
6. HLA-DR specificity assay and epitope mapping
To demonstrate that antigen-binding domains selected from the HuCAL library bound
25 specifically to a binding site on the N-terminal domain of human MHCII receptor
largely conserved between alleles and hitherto unknown in the context of cell killing by
receptor cross linking, we undertook an assessment of their binding specificity, and it
was attempted to characterise the binding epitope.
30 The Fab antibody fragments MS-GPC-8-27-7, MS-GPC-8-27-10, MS-GPC-8-6-13,
MS-GPC-8-27-41 , MS-GPC-8-6-47, MS-GPC-8-10-57, MS-GPC-8-6-27, MS-GPC-8
and MS-GPC-8-6 showed specificity of binding to HLA-DR protein but not to non-
HLA-DR proteins. Fab fragments selected from the HuCAL library were tested for
reactivity with the following antigens: HLA-DR protein (DRA*0101/DRB1*0401;
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prepared as example 1, and a set of unrelated non-HLA-DR proteins consisting of
BSA, testosterone-BSA, lysozyme and human apotransferrin. An empty well (Plastic)
was used as negative control. Coating of the antigen MHCII was performed over night
at 1 ug/well in PBS (Nunc-MaxiSorp TM) whereas for the other antigens (BSA,
5 Testosterone-BSA, Lysozyme, Apotransferrin) 10 ug/well was used. Next day wells
were blocked in 5% non-fat milk for 1 hr followed by incubation of the respective
antibodies (anti-MHCII-Fabs and an unrelated Fab (Mac1-8A)) at 100 ng/well for 1h.
After washing in PBS the anti-human IgG F(ab')2-peroxidase-conjugate at a 1:10000
dilution in TBS (supplemented with 5% w/v non-fat dry-milk/0.05% v/v Tween 20) was
1 0 added to each well for 1 h. Final washes were carried out in PBS followed the addition
the substrate POD (Roche). Color-development was read at 370 nM in an ELISA-
Reader
All anti-HLA-DR antibody fragments MS-GPC-8-27-7, MS-GPC-8-27-10, MS-GPC-8-
15 6-13, MS-GPC-8-27-41 , MS-GPC-8-6-47, MS-GPC-8-10-57, MS-GPC-8-6-27, MS-
GPC-8 and MS-GPC-8-6 demonstrated high specificity for HLA-DR, as evidenced by
the much higher mean fluorescence intensity resulting from incubation of these
antibody fragments with HLA-DR derived antigens compared to controls (Figure 1a).
In a similar experiment, the Fab fragments MS-GPC-1, MS-GPC-6, MS-GPC-8 and
20 MS-GPC-10 were found to bind to both the DRA*0101/DRB1*0401 (preparaed as
above) as well as to a chimeric DR-IE consisting of the N-terminal domains of
DRA*0101 and DRB1*0401 with the remaining molecule derived from a murine
class II homologue lEd (Ito et al., 1996) (Figure 1b).
25 To demonstrate the broad-DR reactivity of anti-HLA-DR antibody fragments and IgGs
of the invention, the scFv forms of MS-GPC-1, 6, 8 and 10, and IgG forms of MS-
GPC-8, MS-GPC-8-10-57, MS-GPC-8-27-51 & MS-GPC-8-6-13 were tested for
reactivity against a panel of Epstein-Barr virus transformed B cell lines obtained from
ECACC (Salisbury UK), each homozygous for one of the most frequent DR alleles in
30 human populations (list of cell lines and alleles shown in Figure 2). The antibody
fragments were also tested for reactivity against a series of L cells transfected to
express human class II isotypes other than DRB1: L105.1, L257.6, L25.4, L256.12 &
L21.3 that express the molecules DRB3*0101, DRB4*0101, DP0103/0402, DP
0202/0201, and DQ0201/0602 respectively (Klohe et al, 1988).
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Reactivity of an antigen-binding fragment to the panel of cell-lines expressing various
MHC- class II molecules was demonstrated using an immunofluorescence procedure
as for example, described by Otten et al (1997). Staining was performed on 2x1 0 5
5 cells using an anti-FLAG M2 antibody as the second reagent against the M2 tag
carried by each anti-HLA-DR antibody fragment and a fluorescein labelled goat anti-
mouse Ig (BD Pharmingen, Torrey Pine, CA, USA) as a staining reagent. Cells were
incubated at 4°C for 60 min with a concentration of 200 nM of the anti-HLA-DR
antibody fragment, followed by the second and third antibody at concentrations
10 determined by the manufacturers. For the IgG form, the second antibody was omitted
and the IgG detected using a FITC-labeled mouse anti-human lgG4 (Serotec, Oxford,
UK) . Cells were washed between incubation steps. Finally the cells were washed and
subjected to analysis using a FACS Calibur (BD Immunocytometry Systems, San
Jose, CA, USA).
15
Figure 2 shows that the scFv-fragments MS-GPC-1, 6, 8 and 10, and IgG forms of
MS-GPC-8, MS-GPC-8-10-57, MS-GPC-8-27-51 & MS-GPC-8-6-13 react with all
DRB1 allotypes tested. This observation taken together with the observation that all
anti-HLA-DR antibody fragments react with chimeric DR-IE, suggests that all selected
20 anti-HLA-DR antibody fragments recognize the extracellular first domain of the
monomorphic DRa chain or a monomorphic epitope on extracellular first domain of
the DR0 chain.
We then attempted to localize the binding domains of MS-GPC-8-10-57 and MS-
25 GPC-8-27-41 further by examining competitive binding with murine antibodies for
which the binding domains on HLA-DR are known. The murine antibodies L243 and
LB3.1 are known to bind to the a1 domain, 1-1C4 and 8D1 to the 01 domain and
10F12 to the 02 domain (Vidovic et al. 1995b). To this end, an assay was developed
wherein a DR-expressing cell line (LG-2) was at first incubated with the lgG4 forms of
30 MS-GPC-8-10-57 or MS-GPC-8-27-41 , the Fab form of MS-GPC-8-10-57 or the Fab
form of GPC 8, and an unrelated control antibody. Subsequently murine antibodies
were added and the murine antibodies were detected. If the binding site of MS-GPC-
8-10-57 or MS-GPC-8-27-41 overlaps with the binding of a murine antibody, then a
reduced detection of the murine antibody is expected.
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Binding of the lgG4 forms of GPC-8-27-41 and MS-GPC-8-10-57 and the Fab form of
MS-GPC-8-10-57 substantially inhibited (mean fluorescence intensity reduced by >
90%) the binding of 1-1 C4 and 8D1, whereas L243, LB3.1 and 10F12 and a control
5 were only marginally affected. The Fab form of MS-GPC-8 reduced binding of 1-1 C4
by ~ 50% (mean fluorescence dropped from 244 to 118), abolished 8D1 binding and
only marginally affected binding of L243, LB3.1 and 10F12 or the control. An
unrelated control antibody had no effect on either binding. Thus, MS-GPC-8-10-57
and MS-GPC-8-27-41 seem to recognise a pi domain epitope that is highly
10 conserved among allelic HLA-DR molecules.
The whole staining procedure was performed on ice. 1x 10 7 cells of the human B-
lymphoblastoid cell line LG-2 was preblocked for 20 Min. in PBS containing 2% FCS
and 35 ug/ml Guinea Pig IgG ("FACS-Buffer"). These cells were divided into 3 equal
parts A, B, and C of approximately 3.3 x 10 6 cells each, and it was added to A.) 35 pg
MS-GPC-8-10-57 or MS-GPC-8-27-41 lgG4, to B.) 35 ug MS-GPC-8-10-57 Fab or
MS-GPC-8 Fab, and to C.) 35 pg of an unrelated lgG4 antibody as negative control,
respectively, and incubated for 90 min. Subsequently A, B, C were divided in 6 equal
parts each containing 5.5 x 10 5 cells, and 2 pg of the following murine antibodies were
added each to one vial and incubated for 30 min: 1.) purified mlgG ; 2.) L243; 3.)
LB3.1; 4.) 1-1 C4; 5.) 8D1; 6.) 10F12. Subsequently, 4ml of PBS were added to each
vial, the vials were centrifuged at 300g for 8 min, and the cell pellet resuspended in 50
pi FACS buffer containing a 1 to 25 dilution of a goat-anti-murine Ig-FITC conjugate at
20 pg/ml final concentration (BD Pharmingen, Torrey Pines, CA, USA). Cells were
incubated light-protected for 30 min. Afterwards, cells were washed with 4 ml PBS,
centrifuged as above and resuspended in 500 pi PBS for analysis in the flow
cytometer (FACS Calibur, BD Immunocytometry Systems, San Jose, CA, USA).
The PepSpot technique (US 6040423; Heiskanen et al., 1999) is used to further
30 identify the binding epitope for MS-GPC 8-10-57. Briefly, an array of 73 overlapping
15mer peptides is synthesised on a cellulose membrane by a solid phase peptide
synthesis spotting method (WO 00/12575). These peptide sequences are derived
from the sequence of the crt and Ii1 domains of HLA-DR4Dw14, HLA-DRA1*0101
(residues 1-81) and HLA-DRB1*0401 (residues 2-92), respectively, and overlap by
50
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20
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two amino acids. Second, such an array is soaked in 0.1% Tween-20/PBS (PBS-T),
blocked with 5% BSA in PBS-T for 3 hours at room temperature and subsequently
washed three times with PBS-T. Third, the prepared array is incubated for 90 minutes
at room temperature with 50 ml of a 5 mg/l solution of the IgG form of GPC-8-10-57 in
5 1% BSA/PBS-T. Fourth, after binding, the membrane is washed three times with
PBS-T and subsequently incubated for 1 hour at room temperature with a goat anti-
human light chain antibody conjugated to horseradish peroxidase diluted 1/5000 in
1% BSA/PBS-T. Finally, the membrane is washed three times with PBS-T and any
binding determined using chemiluminescence detection on X-ray film. As a control for
10 unspecific binding of the goat anti-human light chain antibody, the peptide array is
stripped by the following separate washings each at room temperature for 30 min:
PBS-T (2 times), water, DMF, water, an aequeous solution containing 8M urea, 1%
SDS, 0.5% DTT, a solution of 50% ethanol, 10% acetic acid in water (3 times each)
and, finally, methanol (2 times). The membrane is again blocked, washed, incubated
15 with goat anti-human I light chain antibody conjugated to horseradish peroxidase and
developed as described above.
7. Affinity of anti- HLA-DR antibody and antibody fragments
In order to demonstrate the superior binding properties of anti-HLA antibody
20 fragments of the invention, we measured their binding affinities to the human MHC
class II DR protein (DRA*0101/DRB1*0401) using standard equipment employing
plasmon resonance principles. Surprisingly, we achieved affinities in the sub-
nanomolar range for IgG forms of certain anti-HLA-DR antibody fragments of the
invention. For example, the affinity of the IgG forms of MS-GPC-8-27-41 , MS-GPC-8-
25 6-13 & MS-GPC-8-10-57 was measured as 0.3, 0.5 and 0.6 nM respectively (Table
3a). Also, we observed high affinities in the range of 2-8 nM for Fab fragments affinity
matured at the CDR1 and CDR3 light chain regions (Table 3b). Fab fragments affinity
matured at only the CDR3 light chain region showed affinities in the range of 40 to
100 nM (Table 3c), and even Fab fragments of non-optimised HuCAL antigen binding
30 domains showed affinities in the sub p,M range (Table 3d). Only a moderate increase
in Kon (2-fold) was observed following CDR3 optimisation (Kon remained
approximately constant throughout the antibody optimization process in the order of 1
x 10 5 M"V 1 ), whilst a significant decrease in Koff was a surprising feature of the
optimisation process - sub 100 s" 1 , sub 10 s" 1 , sub 1 s" 1 and sub 0.1 s" 1 for the
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unoptimised Fabs, CDR3 optimised Fabs, CDR3/CDR1 optimised Fabs and IgG
forms of anti-HLA-DR antibody fragments of the invention.
The affinities for anti-HLA antibody fragments of the invention were measured as
5 follows. All measurements were conducted in HBS buffer (20mM HEPES, 150mM
NaCI, pH7.4) at a flow rate of 20ul/min at 25°C on a BIAcore3000 instrument (Biacore
AB, Sweden). MHC class II DR protein (prepared as example 1) was diluted in
100mM sodium acetate pH 4.5 to a concentration of 50-100 mg/ml, and coupled to a
CM5 chip (Biacore AB) using standard EDC-NHS coupling chemistry with subsequent
10 ethanolamine treatment as manufacturers directions. The coating density of MHCII
was adjusted to between 500 and 4000 RU. Affinities were measured by injection of 5
different concentrations of the different antibodies and using the standard software of
the Biacore instrument. Regeneration of the coupled surface was achieved using
10mM glycine pH2.3 and 7.5mM NaOH.
15
8. Multivalent killing activity of anti HLA-DR antibodies and antibody fragments
To demonstrate the effect of valency on cell killing, a cell killing assay was performed
using monovalent, bivalent and multivalent compositions of anti-HLA-DR antibody
fragments of the invention against GRANTA-519 cells. Anti-HLA-DR antibody
20 fragments from the HuCAL library showed much higher cytotoxic activity when cross-
linked to form a bivalent composition (60 - 90% killing at antibody fragment
concentration of 200 nM) by co-incubation with anti-FLAG M2 mAb (Figure 3)
compared to the monovalent form (5 - 30% killing at antibody fragment concentration
of 200 nM). Incubation of cell lines alone or only in the presence of anti-FLAG M2
25 mAb without co-incubation of anti-HLA-DR antibody fragments did not lead to
cytotoxicity as measured by cell viability. Treatment of cells as above but using 50 nM
of the lgG4 forms (naturally bivalent) of the antibody fragments MS-GPC-8, MS-GPC-
8-6-13, MS-GPC-8-10-57 and MS-GPC-8-27-41 without addition of anti-FLAG M2
mAb showed a killing efficiency after 4 hour incubation of 76%, 78%, 78% and 73%
30 respectively.
Furthermore, we observed that higher order valences of the anti-HLA-DR antibody
fragments further decrease cell viability significantly. On addition of Protein G to the
incubation mix containing the IgG form of the anti-HLA-DR antibody fragments, the
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multivalent complexes thus formed further decrease cell viability compared to the
bivalent composition formed from incubation of the anti-HLA-DR antibody fragments
with only the bivalent IgG form.
5 The killing efficiency of anti-HLA-DR antibody fragments selected from the HuCAL
library was tested on the HLA-DR positive tumor cell line GRANTA-519 (DSMZ,
Germany). 2x1 0 5 cells were incubated for 4 h at 37°C under 6% C0 2 with 200 nM
anti-HLA-DR antibody fragments in RPMI 1640 (PAA, Germany) supplemented with
2,5% heat inactivated FBS (Biowhittaker Europe, BE), 2mM L-glutamine, 1% non-
10 essential amino acids, 1 mM sodium pyruvate and 0,1 mg/ml kanamycin. Each anti-
HLA-DR antibody fragment was tested for its ability to kill activated tumor cells as a
monovalent anti-HLA-DR antibody fragment or as a bivalent composition by the
addition of 100 nM of a bivalent cross-linking anti-FLAG M2 mAb. After 4 h incubation
at 37°C under 6% C0 2 , cell viability was determined by trypan blue staining and
15 subsequent counting of remaining viable cells (Current Protocols in Immunology,
1997).
The above experiment was repeated using KARPAS-422cells against a multivalent
form of IgG forms of MS-GPC-8-1 0-57 and MS-GPC-8-27-41 prepared by a pre-
20 incubation with a dilution series of the bacterial protein Protein G. Protein G has a
high affinity and two binding sites for IgG antibodies, effectively cross-linking them to
yield a total binding valency of 4. In a control using IgG alone without preincubation
with Protein G, approximately 55% of cells were killed, while cell killing using IgG pre-
incubated with Protein G gave a maximum of approximately 75% at a molar ratio of
25 IgG antibody/Protein G of ~ 6 (based on a molecular weight of Protein G of 28.5 kD).
Higher or lower molar ratios of IgG antibody/Protein G approached the cell killing
efficiency of the pure IgG antibodies.
9. Killing efficiency of anti-HLA-DR antibody fragments
30 Experiments to determine the killing efficiency of the anti-HLA-DR cross-linked
antibody fragments against other tumor cell lines that express HLA-DR molecules
were conducted analogous to example 8. Tumor cell lines that show greater than 50%
cell killing with the cross linked Fab form of MS-GPC-8 after 4 h incubation include
MHH-CALL4, MN 60, BJAB, BONNA-12 which represent the diseases B cell acute
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lymphoid leukemia, B cell acute lymphoid leukemia, Burkitt lymphoma and hairy cell
leukemia respectively. Use of the cross-linked Fab form of the anti-HLA-DR antibody
fragments MS-GPC-1, 6 and 10 also shows similar cytotoxic activity to the above
tumor cell lines when formed as a bivalent agent using the cross-linking anti-FLAG
5 M2 mAb.
The method described in example 8 was used to determine the maximum killing
capacity for each of the cross-linked bivalent anti-HLA-DR antibody fragments against
Priess cells. The maximum killing capacity observed for MS-GPC-1 , MS-GPC-6, MS-
10 GPC-8 & MS-GPC-1 0 was measured as 83%, 88%, 84% and 88% respectively.
Antibody fragments generated according to example 4, when cross linked using anti-
FLAG M2 mAb as above, also showed improved killing ability against GRANTA and
Priess cells (Table 4).
15 10. Killing efficiency of anti-HLA-DR IgG antibodies of human composition
Compared to corresponding murine antibodies (Vidovic et al, 1995b; Nagy & Vidovic,
1996; Vidovic & Toral; 1998), we were surprised to observe significantly improved
killing efficiency of IgG forms of certain anti-HLA-DR antibody fragments of the
invention (Table 5). Following the method described in examples 8 and 9 but at 50
20 nM, repeated measurements (3 to 5 replica experiments where cell number was
counted in duplicate for each experiment) were made of the killing efficiency of the
IgG forms of certain antibody fragments of the invention. When applied at a final
concentration of only 50 nM, IgGs of the antibody fragments MS-GPC-8, MS-GPC-8-
6-13, MS-GPC-8-10-57 & MS-GPC-8-27-41 killed more than 50% of cells from 16, 22,
25 19 and 20 respectively of a panel of 24 human tumor cell lines that express HLA-DR
antigen at a level greater than 10 fluorescent units as determined by example 11.
Cells were treated with the two murine anti-HLA-DR antibodies L243 (Vidovic et al,
1995b) and 8D1 (Vidovic & Toral; 1998) at a significantly higher final concentration of
mAb (200 nM), which reduced cell viability to a level below 50% viable cells in only 13
30 and 12 of the 24 HLA-DR expressing cells lines, respectively. The cell line MHH-
PREB-1 was singled out and not accounted as part of the panel of 24 cell lines
despite its expression of HLA-DR antigen at a level greater than 10 fluorescent units
due to the inability of any of the above antibodies to induce any significant reduction
of cell viability. This is further explained in example 12.
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Indeed, even at the significantly increased concentration, the two murine antibodies
treated at 200 nM showed significantly less efficient killing compared to the IgG forms
of anti-HLA DR antibody fragments of the invention. Not only do IgG forms of the
5 human anti-HLA-DR antibody fragments of the invention show an overall increase in
cell killing at lower concentrations compared to the murine antibodies, but they show
less variance in killing efficiency across different cell lines.The coefficient of variance
in killing for the human antibodies in this example is 32% (mean %killing = 68 +/- 22%
(SD)), compared to over 62% (mean %killing = 49 +/- 31% (SD)) for the mouse
10 antibodies. Statistically controlling for the effect on killing efficiency due to HLA
expression by fitting logistic regression models to mean percentage killing against
log(mean HLA DR expression) supports this observation (Figure 4). Not only is the
fitted curve for the murine antibodies consitently leower than that for the human, but a
larger variance in residuals from the murine antibody data (SD = 28%) is seen
15 compared to the variance in residuals from the human antibody data (16%).
11. Killing selectivity of antigen-binding domains against a human antigen for
activated versus non-activated cells
Human peripheral B cells were used to demonstrate that human anti-HLA-DR mAb-
20 mediated cell killing is dependent on cell-activation. 50 ml of heparinised venous
blood was taken from an HLA-DR typed healthy donor and fresh peripheral blood
mononuclear cells (PBMC) were isolated by Ficoll-Hypaque Gradient Centrifugation
(Histopaque-1077; Sigma) as described, in Current Protocols in Immunology (John
Wiley & Sons, Inc.; 1999). Purified B cells (-5% of peripheral blood leukocytes) were
25 obtained from around 5x1 0 7 PBMC using the B-cell isolation kit and MACS LS + A/S +
columns (Miltenyi Biotec, Germany) according to manufacturers guidelines.
Successful depletion of non-B cells was verified by FACS analysis of an aliquot of
isolated B cells (HLA-DR positive and CD19 positive). Double staining and analysis is
done with commercially available antibodies (BD Immunocytometry Systems, San
30 Jose, CA, USA) using standard procedures as for example described in Current
Protocols in Immunology (John Wiley & Sons, Inc.; 1999). An aliquot of the isolated B
cells was tested for the ability of the cells to be activated by stimulation with
Pokeweed mitogen (PWM) (Gibco BRL, Cat. No. 15360-019) diluted 1:25 in RPMI
1640 (PAA, Germany) supplemented with 10% FCS (Biowhittaker Europe, BE), 2mM
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L-glutamine, 1% non-essential amino acids, 1mM sodium pyruvate and 0,1mg/ml
kanamycin by incubation at 37°C under 6% C0 2 for three days. Successful activation
was verified by FACS analysis of HLA-DR expression on the cell surface (Current
Protocols in Immunology, John Wiley & Sons, Inc.; 1999).
5
The selectivity for killing of activated cells versus non-activated cells was
demonstrated by incubating 1x10 6 /ml B cells activated as above compared to non-
activated cells, respectively with 50 nM of the IgG forms of MS-GPC-8-10-57, MS-
GPC-8-27-41 or the murine IgG 10F12 (Vidovic et a!., 1995b) in the medium
10 described above but supplemented with 2,5% heat inactivated FCS instead of 10%,
or with medium alone. After incubation at 37°C under 6% C0 2 for 1 or 4h, cell viability
was determined by fluorescein diacetate staining (FDA) of viable and propidium
iodide staining (PI) of dead cells and subsequent counting of the green (FDA) and red
(PI) fluorescent cells using a fluorescence microscope (Leica, Germany) using
15 standard procedures (Current Protocols in Immunology, 1997).
B cell activation was shown to be necessary for cell killing. In non-activated cells after
1 h of incubation with the anti-HLA-DR antibodies, the number of viable cells in the
media corresponded to 81%, 117% 126% and 96% of the pre-incubation cell density
20 for MS-GPC-8-10-57 (IgG), MS-GPC-8-27-41 (IgG), 10F12 and medium alone,
respectively. In contrast, the number of viable activated B cells after 1 h incubation
corresponded to 23%, 42% 83% and 66% of the pre-incubation cell density for MS-
GPC-8-10-57 (IgG), MS-GPC-8-27-41 (IgG), 10F12 and medium alone, respectively.
After 4 h of incubation, 78%, 83% 95% and 97% of the pre-incubation cell density for
25 MS-GPC-8-10-57 (IgG), MS-GPC-8-27-41 (IgG), 10F12 and medium alone were
found viable in non-activated cells, whereas the cell density had dropped to 23%, 24%
53% and 67% of the pre-incubation cell density for MS-GPC-8-10-57 (IgG), MS-GPC-
8-27-41 (IgG), 10F12 and medium alone, respectively, in activated cells.
30 12. Killing activity of anti-HLA antibody fragments against the cell line MHH PreB 1
As evidenced in Table 5, we observed that our cross-linked anti-HLA-DR antibody
fragments or IgGs did not readily kill a particular tumor cell line expressing HLA-DR
at significant levels. We hypothesized that although established as a stable cell line,
cells in this culture were not sufficiently activated. Therefore, we conducted an
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experiment to stimulate activity of the MHH preB1 cell line, using increased cell-
surface expression of HLA-DR molecule as a marker of activation as follows.
Non-ad herently growing MHH preB1 cells were cultivated in RPMI medium containing
5 the following additives (all from Gibco BRL and Bio Whittaker): 1 0% FCS, 2 mM
L-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate and 1x
Kanamycin. Aliquots were activated to increase expression of HLA-DR molecule by
incubation for one day with Lipopolysaccharide (LPS, 10 ug/ml), Interferon-gamma
(IFN-y, Roche, 40 ng/ml) and phyto-hemagglutinin (PHA, 5 ug/ml). The cell surface
10 expression of HLA-DR molecules was monitored by flow cytometry with the FITC-
conjugated mAb L243 (BD Immunocytometry Systems, San Jose, CA, USA).
Incubation of MHH preB1 for one day in the presence of LPS, IFN- y and PHA
resulted in a 2-fold increase in HLA-DR surface density (mean fluorescence shift
from 190 to 390). Cell killing was performed for 4 h in the above medium but
15 containing a reduced FCS concentration (2.5%). A concentration series of the IgG
forms of MS-GPC-8-27-41 & MS-GPC-8-10-57 was employed, consisting of final
antibody concentrations of 3300, 550, 92, 15, 2.5, 0.42 and 0.07 nM, on each of an
aliquot of non-activated and activated cells. Viable cells were identified
microscopically by exclusion of Trypan blue. Whereas un-activated cell viability
20 remains unaffected by the antibody up to the highest antibody concentration used,
cell viability is dramatically reduced with increasing antibody concentration in
activated MHH PreB1 cells (Figure 5).
13. Killing efficiency of anti-HLA-DR IgG antibodies of human composition against ex-
25 vivo chronic lymphoid leukemia cells
Using B cells isolated and purified from 10 patients suffering from chronic lymphoid
leukemia (CLL), we demonstrated that IgG forms of anti-HLA-DR antibody fragments
of the invention showed efficacy in killing of clinically relevant cells using an ex-vivo
assay. B-cells were isolated and purified from 10 unrelated patients suffering from
30 CLL (samples kindly provided by Prof Hallek, Ludwig Maximillian University, Munich)
according to standard procedures (Buhmann et al., (1999)). 2x1 0 5 cells were treated
with 100 nM of IgG forms of the anti-HLA-DR antibody fragments MS-GPC-8, MS-
GPC-8-10-57 or MS-GPC-8-27-41 and incubated for 4 or 24 hours analogous to
examples 8 and 9. A replica set of cell cultures was established and activated by
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incubation with HeLa-cells expressing CD40 ligand on their surface for three days
before treatment with antibody (Buhmann et al., 1999). As controls, the murine IgG
10F12 (Vidovic et al., 1995b) or no antibody was used. Cell viability for each
experiment was determined as described in example 12.
5
Surprisingly, IgG forms of the anti-HLA-DR antibody fragments of the invention
showed highly efficient and uniform killing - even across this diverse set of patient
material. After only 4 hours of treatment, all three human IgGs gave a significant
reduction in cell viability compared to the controls, and after 24 hours only 33% of
10 cells remained viability (Figure 6). We found that on stimulating the ex-vivo cells
further according to Buhmann et al (1999), the rate of killing was increased such that
after only 4 hours culture with the human antibodies, only 24% of cells remained
viable on average for all patient samples and antibody fragments of the invention.
15 14. Determination of EC50 for anti-HLA-DR antibody fragments
We demonstrated superior Effective Concentration at 50% effect (EC50) values in a
cell-killing assay for certain forms of anti-HLA-DR antibody fragments selected from
the HuCAL library compared to cytotoxic murine anti-HLA-DR antibodies (Table 6).
20 The EC50 for anti-HLA-DR antibody fragments selected from the HuCAL library were
estimated using the HLA-DR positive cell line PRIESS or LG2 (ECACC, Salisbury
UK). 2x1 0 5 cells were incubated for 4 h at 37°C under 6% C0 2 in RPMI 1640 (PAA,
Germany) supplemented with 2,5% heat inactivated FBS (Biowhittaker Europe, BE),
2mM L-glutamine, 1% non-essential amino acids, 1mM sodium pyruvate and
25 0,1mg/ml kanamycin, together with dilution series of bivalent anti-HLA-DR antibody
fragments. For the dilution series of Fab antibody fragments, an appropriate
concentration of Fab fragment and anti-FLAG M2 antibody were premixed to
generate bivalent compositions of the anti-HLA-DR antibody fragments. The
concentrations stated refer to the concentration of bivalent composition such that the
30 IgG and Fab EC50 values can be compared.
After 4 h incubation with bivalent antibody fragments at 37°C under 6% C0 2 , cell
viability was determined by fluorescein diacetate staining and subsequent counting of
remaining viable cells (Current Protocols in Immunology, 1997). Using standard
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statistical software, non-linear logistic regression curves were fitted to replica data
points and the EC50 estimated for each antibody fragment.
When cross-linked using the anti-FLAG M2 antibody, the Fab fragments MS-GPC-1 ,
5 MS-GPC-8 & MS-GPC-1 0 selected from the HuCAL library (Example 4) showed an
EC50 of less than 120 nM as expressed in terms of the concentration of the
monovalent fragments, which corresponds to a 60 nM EC50 for the bivalent cross-
linked (Fab)dimer-anti-Flag M2 conjugate. (Figure 7a). When cross-linked using the
anti-FLAG M2 antibody, anti-HLA-DR antibody fragments optimised for affinity within
10 the CDR3 region (Example 4) showed a further improved EC50 of less than 50 nM, or
25 nM in terms of the bivalent cross-linked fragment (Figure 7b), and those
additionally optimised for affinity within the CDR1 region showed an EC50 of less than
30 nM (15 nM for bivalent fragment). In comparison, the EC50 of the cytotoxic murine
anti-HLA-DR antibodies 8D1 (Vidovic & Toral; 1998) and L243 (Vidovic et al; 1995b)
15 showed an EC50 of over 30 and 40 nM, respectively, within the same assay (Figure
7c).
Surprisingly, the IgG form of certain antibody fragments of the invention showed
approximately 1 .5 orders of magnitude improvement in EC50 compared to the murine
20 antibodies (Figure 7d). For example, the IgG forms of MS-GPC-8-1 0-57 & MS-GPC-
8-27-41 showed an EC50 of 1 .2 and 1 .2 nM respectively. Furthermore, despite being
un-optimised for affinity, the IgG form of MS-GPC-8 showed an EC50 of less than 10
nM.
25 As has been shown in examples 11 and 12, the efficiency of killing of un-activated
cells (normal peripheral B and MHH PreB cells respectively) is very low. After
treatment with 50 nM of the IgG forms of MS-GPC-8-1 0-57 & MS-GPC-8-27-41, 78%
and 83% of normal peripheral B cells, respectively, remain viable after 4 hours.
Furthermore, at only 50nM concentration or either IgG, virtually 100% viability is seen
30 for MHH PreB1 cells. Indeed, a decrease in the level of viability to below 50% cannot
be achieved with these un-activated cells using reasonable concentration ranges (0.1
to 300 nM) of IgG or bivalent cross-linked Fab forms of the anti-HLA DR antibody
fragments of the invention. Therefore, the EC50 for these un-activated cell types can
be estimated to be at least 5 times higher than that shown for the non-optimised Fab
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forms (EC50 ~ 60 nM with respect to cross-linked bivalent fragment), and at least 10
times and 100 times higher than EC50s shown for the VHCDR3 optimised Fabs (~ 25
nM with respect to cross-linked bivalent fragment) and IgG forms of MS-GPC-8-10-57
(~1 .2 nM) & MS-GPC-8-27-41 (~1 .2 nM) respectively.
5
15. Mechanism of cell-killing
The examples described above show that cell death occurs - needing only certain
multivalent anti-HLA-DR antibody fragments to cause killing of activated cells. No
further cytotoxic entities or immunological mechanisms were needed to cause cell
10 death, therefore demonstrating that cell death is mediated through an innate pre-
programmed mechanism of the activated cell. The mechanism of apoptosis is a
widely understood process of pre-programmed cell death. We were surprised by
certain characteristics of the cell killing we observed that suggested the mechanism of
killing for activated cells when exposed to our human anti-HLA-DR antibody
15 fragments was not what is commonly understood in the art as "apoptosis". For
example, the observed rate of cell killing appeared to be significantly greater than the
rate reported for apoptosis of immune cells (about 10 - 15 h; Truman et al., 1994).
Two experiments were conducted to demonstrate that the mechanism of cell killing
proceeded by a non-apoptotic mechanism.
20
First, we used Annexin-V-FITC and propidium iodide (PI) staining techniques to
distinguish between apoptotic and non-apoptotic cell death - cells undergoing
apoptosis, "apoptotic cells", (Annexin-V positive/PI negative) can be distinguished
from necrotic ("Dead") (Annexin-V positive/PI positive) and fully functional cells
25 (Annexin-V negative/PI negative). Using the procedures recommended by the
manufacturers of the AnnexinV and PI assays, 1x10 6 /ml Priess cells were incubated
at 37°C under 6% C0 2 with or without 200 nM anti-HLA-DR antibody fragment MS-
GPC-8 together with 100 nM of the cross-linking anti-FLAG M2 mAb in RPMI 1640
(PAA, DE) supplemented with 2,5% heat inactivated FCS (Biowhittaker Europe, BE),
30 2mM L-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate and 0,1
mg/ml kanamycin. To provide an apoptotic cell culture as control, 1x10 6 /ml Priess
cells were induced to enter apoptosis by incubation in the above medium at 37°C
under 6% C0 2 with 50 (j,g/ml of the apoptosis-inducing anti-CD95 mAb DX2 (BD
Pharmingen, Torrey Pine, CA, USA) cross-linked with 10 jwg/ml Protein-G. At various
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incubation times (1,15 and 60 min, 3 and 5 h) 200 samples were taken, washed
twice and stained with Annexin-V-FITC (BD Pharmingen, Torrey Pine, CA, USA) and
PI using Annexin-V binding buffer following the manufacturer's protocol. The amount
of staining with Annexin-V-FITC and PI for each group of cells is analysed with a
5 FACS Calibur (BD Immunocytometry Systems, San Jose, CA, USA).
Cell death induced through the cross-linked anti-HLA-DR antibody fragments shows a
significantly different pattern of cell death than that of the anti-CD95 apoptosis
inducing antibody or the cell culture incubated with anti-FLAG M2 mAb alone. The
10 percentage of dead cells (as measured by Annexin-V positive/PI positive staining) for
the anti-HLA-DR antibody fragment/anti-FLAG M2 mAb treated cells increases far
more rapidly than that of the anti-CD95 or the control cells (Figure 8a). In contrast, the
percentage of apoptotic cells (as measured by Annexin-V positive/PI negative
staining) increases more rapidly for the anti-CD95 treated cells compared to the
15 cross-linked anti-HLA-DR antibody fragments or the control cells (Figure 8b).
Second, we inhibited caspase activity using zDEVD-fmk, an irreversible Caspase-3
inhibitor, and zVAD-fmk, a broad spectrum Caspase inhibitor (both obtained from
BioRad, Munich, DE). The mechanism of apoptosis is characterized by activity of
20 caspases, and we hypothesized that if caspases were not necessary for anti HLA-DR
mediated cell death, we would observe no change in the viability of cells undergoing
cell death in the presence of these caspase inhibitors compared to those without.
2x1 0 5 Priess cells were preincubated for 3 h at 37°C under 6% C0 2 with serial
dilutions of the two caspase inhibitors ranging from 180 |xM to 10 mM in RPMI 1640
25 (PAA, DE) supplemented with 2,5% heat inactivated FCS (Biowhittaker Europe, BE),
2m M L-glutamine, 1% non-essential amino acids, 1mM sodium pyruvate and
0,1mg/ml kanamycin. HLA-DR mediated cell death was induced by adding 200 nM of
the human anti-HLA-DR antibody fragment MS-GPC-8 and 100 nM of the cross-
linking anti-M2 mAb. An anti-CD95 induced apoptotic cell culture served as a control
30 for the activity of inhibitors (Drenou et al., 1999). After further incubation at 37°C and
6% C0 2 , cell viability after 4 and 24 h was determined by trypan blue staining and
subsequent counting of non-stained cells. As we expected, cell viability of the anti-
HLA-DR treated cell culture was not significantly modified by the presence of the
Caspase inhibitors, while cell death induced through anti-CD95 treatment was
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significantly decreased for the cell culture pre-incubated with the Caspase inhibitors.
This observation supports our hypothesis that HLA-DR mediated cell death proceeds
through a non-apoptotic mechanism that is independent of caspase proteases that
can be inhibited by zDEVD-fm or zVAD-fmk.
5
76. In vivo therapy for cancer using an HLA-DR specific antibody
We demonstrate that antigen-binding domains of human composition can
successfully be used as a therapeutic for the treatment of cancer.
Immunocompromised mice - such as scid, nude or Rag-1 knockout - are inoculated
10 with a DR+ human lymphoma or leukemia cell line of interest. The tumor cell dose,
usually 1x1 0 6 to 1x10 7 /mouse, is established for each tumor tested and administered
subcutaneously (s.c.) or intravenously (i.v.). The mice are treated i.v. or s.c with the
IgG form of the anti-HLA-DR antibody fragments MS-GPC-8, MS-GPC-8-10-57, MS-
GPC-8-27-41 or others of the invention prepared as described above, using doses of
15 1 to 25 mg/kg over 5 days. Survival of. anti-HLA-DR treated and control untreated
mice is monitored for up to 8 weeks after cessation of treatment. Tumor progression
in the mice inoculated s.c. is additionally quantified by measuring tumor surface area.
Significant prolongation of survival of up to 80% of anti-HLA-DR treated mice is
observed during the experiment, and up to 50% mice survive at the end of the
20 experiment. In s.c. inoculated and untreated mice, the tumor reaches a surface area
of 2 - 3 cm 2 , while in anti-HLA-DR treated animals the tumor surface area is
significantly less.
17. Immunosuppression using anti-HLA-DR antibody fragments measured by
25 reduction in IL-2 secretion
We were surprised to observe that certain anti-HLA DR antibody fragments of the
invention displayed substantial immunomodulatory properties within an assay
measuring IL-2 secretion from immortalized T-cells. IgG forms of the antibody
fragments MS-GPC-8-6-13, MS-GPC-8-10-57 & MS-GPC-8-27-41 showed very
30 strong immunosuppressive properties in this assay with sub-nanomolar IC50 values
and virtually 100% maximal inhibition (Figure 9a). Particularly surprising was our
observation that certain monvalent compositions of the antibody fragments of the
invention were able to strongly inhibit IL-2 secretion in the same assay. For example,
Fab forms of the VHCDR3-selected and VLCDR3A/LCDR1 optimised antibody
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fragments showed low single-digit nano-M IC50s and also almost 100% maximal
inhibition (Figure 9b). Other monvalent anti-HLA DR antibody fragments of the
invention showed significant immunosuppressive properties in the assay compared to
control IgG and Fab fragments (Table 7).
5
The immunomodulatory properties of anti-HLA DR antibody fragments was
investigated by measuring IL-2 secretion from the hybridoma cell line T-Hyb 1
stimulated using DR-transgenic antigen presenting cells (APC) under conditions of
half-maximal antigen stimulation. IL-2 secretion was detected and measured using a
10 standard ELISA method provided by the OptiEIA mouse IL-2 kit of Pharmingen
(Torrey Pine, CA, USA). APCs were isolated from the spleen of unimmunized
chimeric 0401 -IE transgenic mice (Ito et al. 1996) according to standard procedures.
1.5x10 5 APCs were added to 0.2 ml wells of 96-well in RPMI medium containing the
following additives (all from Gibco BRL and PAA): 10 % FCS, 2mM L-glutamine, 1%
15 non-essential amino acids, 1 mM sodium pyruvate and 0.1 g/l kanamycin. Hen egg
ovalbumin was added to a final concentration of 200 pg/ml in a final volume of 100 ul
of the above medium, the cells incubated with this antigen for 30 min at 37°C under
6% C0 2 . Anti-HLA DR antibody fragments were added to each well at various
concentrations (typically in a range from 0.1 to 200 nM), the plate incubated for 1 h at
20 37°C/6% C0 2 and 2x1 0 5 T-Hyb 1 cells added to give a final volume of 200 ul in the
above medium. After incubation for 24 h, 100 pi of supernatant was transferred to an
ELISA plate (Nunc-lmmuno Plate MaxiSorp surface, Nunc, Roskilde, DK) previously
coated with IL-2 Capture Antibody (BD Pharmingen, Torrey Pine, CA, USA), the
amount of IL-2 was quantified according to the manufacturer's directions using the
25 OptiEIA Mouse IL-2 kit and the plate read using a Victor V reader (Wallac, Finland).
Secreted IL-2 in pg/ml was calibrated using the IL-2 standards provided in the kit.
The T-cell hybridoma line T-Hyb1 was established by fusion of a T-cell receptor
negative variant of the thymoma line BW 5147 (ATCC) and lymph node cells from
30 chimeric 0401 -IE transgenic mice previously immunized with hen egg ovalbumin (Ito
et al. 1996). The clone T-Hyb1 was selected for the assay since it responded to
antigen specific stimulation with high IL-2 secretion.
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18. Immunosuppression using an HLA-DR specific antibody measured by T ceil
proliferation
Immunomodulatory properties of the anti-HLA DR antibody fragments were also seen
within an assay that measures T cell proliferation. The IC50 value for inhibition of T
5 cell proliferation of the IgG form of MS-GPC-8-10-57 and MS-GPC-8-27-41 were 11
and 20 nM respectively (Figure 10). The anti-HLA DR antibody fragments were tested
as follows to inhibit the proliferative T cell response of antigen-primed lymph node
cells from mice carrying a chimeric mouse-human class II transgene with an RA-
associated peptide binding site, and lack murine class II molecules (Muller et al.,
10 1990; Woods et al., 1994; Current Protocols in Immunology, Vol. 2, 7.21; Ito et al.,
1996). Here, the immunization takes place in vivo, but the inhibition and readout are
ex vivo. Transgenic mice expressing MHC class II molecules with binding sites of the
RA associated molecule, DRB*0401 were commercially obtained. These mice lack
murine MHC class II, and thus, all Th responses are channelled through a single
15 human RA-associated MHC class II molecule (Ito et al. 1996). These transgenic mice
represent a model for testing human class II antagonists.
The inhibitory effect of the anti-HLA-DR antibody fragments and their IgG forms were
tested on T-cell proliferation measured using chimeric T-cells and antigen presenting
20 cells isolated from the lymph nodes of chimeric 0401 -IE transgenic mice (Taconic,
USA) previously immunized with hen egg ovalbumin (Ito et al. 1996) according to
standard procedures. 1.5x10 5 cells are incubated in 0.2 ml wells of 96-well tissue
culture plates in the presence of ovalbumin (30 ug per well - half-maximal stimulatory
concentration) and a dilution series of the anti-HLA DR antibody fragment or IgG form
25 under test (0.1 nM - 200 nM) in serum free HL-1 medium containing 2 mM L-
glutamine and 0.1 g/l Kanamycin for three days. Antigen specific proliferation is
measured by 3 H-methyl-thymidin(1 uCi/well) incorporation during the last 16h of
culture (Falcioni et al., 1999). Cells are harvested, and 3 H incorporation measured
using a scintillation counter (TopCount, Wallac Finland). Inhibition of T-cell
30 proliferation on treatment with the anti-HLA DR antibody fragment and its IgG form
may be observed by comparison to control wells containing antigen.
64
WO 01/87337
PCT/US01/15625
1 9. Selection of useful polypeptide for the treatment of cancers
In order to select the most appropriate protein/peptide to enter further experiments
and to assess its suitability for use in a therapeutic composition for the treatment of
cancers, additional data are collected. Such data for each IgG form of the anti-HLA
5 antigen antibody fragments can include the binding affinity, in vitro killing efficiency as
measured by EC50 and cytotoxicity across a panel of tumor cell lines, the maximal
percentage cell killing as estimated in vitro, and tumor reduction data and mouse
survival data from in vivo animal models.
10 The IgG form of the anti-HLA antigen antibody fragments that shows the highest
affinity, the lowest EC50 for killing, the highest maximal percentage cell killing and
broadest across various tumor cell lines, the best tumor reduction data and/or the
best mouse-survival data may be chosen to enter further experiments. Such
experiments may include, for example, therapeutic profiling and toxicology in animals
15 and phase I clinical trials in humans.
20. Selection of useful polypeptide for the treatment of diseases of the immune
system
In order to select the most appropriate protein/peptide to enter further experiments
20 and to assess its suitability for use in a therapeutic composition for the treatment of
diseases of the immune system, additional data are collected. Such data for each
monovalent antibody fragment or IgG form of the anti-HLA antigen antibody
fragments can include the affinity, reactivity, specificity, IC50-values, for inhibition of
IL-2 secretion and of T-cell proliferation, or in vitro killing efficiency as measured by
25 EC50 and the maximal percentage cell killing as estimated in vitro, and DR-
transgenic models of transplant rejection and graft vs. host disease.
The antibody fragment or IgG form of the anti-HLA antigen antibody fragments that
shows the lowest EC50, highest affinity, highest killing, best specificity and/or greatest
30 inhibition of T-cell proliferation or IL-2 secretion, and high efficacy in inhibiting
transplant rejection and/or graft vs. host disease in appropriate models, might be
chosen to enter further experiments. Such experiments may include, for example,
therapeutic profiling and toxicology in animals and phase I clinical trials in humans.
65
WO 01/87337
PCT/US01/15625
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67
WO 01/87337
PCT/US01/15625
L-CDR1
SGSSSNIGSNYVS
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Fab
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optimisation
Parental Fab
L-CDR3-optim.
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L-CDR3+1-opt.
68
WO 01/87337 PCT/US01/15625
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69
WO 01/87337
PCT/US01/15625
Table 3a
Affinities of selected lgG4 monoclonal antibodies constructed from F a b's. Errors represent
standard deviations
Binder (lgG 4 )
k on [IvT 1 s^xlO 5
koffts" 1 ] x10" b
K D [nM]
MS-GPC-8-27-41
1.1 ±0.2
3,1 ±0.4
0,31 ± 0.06
MS-GPC-8-6-13
0,7 ±0.1
3± 1
0,5 ±0.2
MS-GPC-8-10-57
0,7 ± 0.2
4± 1
0,6 ±0.2
Table 3b
Affinities of binders obtained out of affinity maturation of CDR1 light chain optimisation
following CDR3 heavy chain optimisation. Errors represent standard deviations
Binder (F ab )
kontM'V 1 ] x10 5
k off [s-''] x10" 3
K D [nM]
MS-GPC-8-6-2
1.2 ± 0.1
0.94 ± 0.07
7.6 ±0.3
MS-GPC-8-6-19
1.1 ±0.1
1.0 ±0.2
9± 1
MS-GPC-8-6-27
1.8 ±0.2
1.1 ± 0.2
6.3 ± 0.6
MS-GPC-8-6-45
1 .20 ± 0.07
1 .03 ± 0.04
8.6 ±0.6
MS-GPC-8-6-13
1.9 ±0.3
0.55 ± 0.05
3.0 ±0.5
MS-GPC-8-6-47
2.0 ± 0.3
0.62 ± 0.04
3.2 ±0.3
MS-GPC-8-10-57
1.7 ±0.2
0.44 ± 0.06
2.7 + 0.3
MS-GPC-8-27-7
1.7 ±0.2
0.57 ± 0.07
3.3 ±0.3
MS-GPC-8-27-10
1 .8 ± 0.2
0.53 ± 0.05
3.0 ±0.2
MS-GPC-8-27-41
1.7 ±0.2
0.49 ± 0.03
2.9 ±0.3
70
WO 01/87337 PCT/US01/15625
Table 3c
Binders obtained out of affinity maturation of GPC8 by CDR3 light chain optimisation
Binder (F a b)
k on [M"V] x10 a
kofflS 1 ] x10' 3
K D [nM]
MS-GPC 8-18
1.06
8.3
78.3
MS-GPC 8-9
1.85
16.6
90.1
MS-GPC 8-1
1.93
20.9
108
MS-GPC 8-17
1.0
5.48
54.7
MS-GPC-8-6 a>
1.2+/- 0.1
5.5 +/- 0.7
8 +/- 12
Chip density 4000
RU MHCII
a) For MS-GPC-8-6 mean and standard deviation of 3 different preparations on 3 different
chips (500, 4000, 3000RU) is shown.
Table 3d
Binders obtained out of HuCAL in scFv form and their converted Fabs
Binder
scF v
Fab
kon [M-V]
x10 5
koff [S-]
x10 -3
K D [nM]
kon [M-V]
x10 5
k 0 ff [s 1 ]
X10" 3
K D [nM]
MS-GPC 1
0.413
61
1500
0.639
53
820
MS-GPC 6
0.435
200
4600
0.135
114
8470 (1 curve)
MS-GPC 8
0.114
76
560
0.99
+/- 0.40
29.0
+/- 8.4
346 a)
+/- 141
MS-GPC 10
0.187
180
9625 .
0.22
63
2860
Chip density 500RU MHCII
a) Affinity data of MS-GPC-8 are based on 8 different Fab-preparations which were
measured on 4 different chips (2 x 500, 1000, 4000RU) and are shown with standard
deviation.
71
WO 01/87337
PCT/US01/15625
Table 4
Killing efficiency after 4 hour incubation of cells with cross-linked anti-HLA-DR antibody
fragments, and maximum killing after 24 hour incubation
Cross-linked Fab fragment
Killing efficiency against
GRANTA
Maximum killing against
Priess
MS-GPC-1
+
+
MS-GPC-6
+
+
MS-GPC-8
+
+
MS-GPC-1 0
+
+
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++
++
MS-GPC-8-17
++
++
MS-GPC-8-6-13
+++
+++
MS-GPC-8-10-57
+++
+++
MS-GPC-8-27-41
+++
+++
72
WO 01/87337
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hairy cell leuk.
hairy cell leuk.
CML
plasma cell leu.
AML (eosinophil)
multiple myeloma
multiple myeloma
B cell non-Hodgkin
B cell precursor leu.
multiple myeloma
AML
AML
CML
HDLM-2
HD-MY-Z
KM-H2
BONNA-12
HC-1
NALM-1
L-363
EOL-1
LP-1
RPMI-8226
MHH-PREB-1
MHH-CALL-2
OPM-2
KASUMI-1
HL-60
LAMA-84
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Table 6
EC50 values for certain anti-HLA-DR antibody fragments of the invention in a cell-killing
assay against lymphoid tumor cells. All EC50 refer to nanomolar concentrations of the
bivalent agent (IgG or cross-linked Fab) such that values for cross-linked Fab and IgG
forms can be compared.
Antibody fragment
Form
Cell line tested
EC50 of cell killing (nM) +/- SE for
bivalent agent
IVIo-CDrO-1
f-ab
nniroo
rRltzoo
54 ± 14
IVlo-orU-o
hab
PRIESS
31+9
Mo-Q^rU-IU
Fab
PRIESS
33 + 5
i\yio dp o h ~7
Mo-orU-o-1 (
Fab
PRIESS
16 + 4
MS-GPC-8-6-2
Fab
PRIESS
8±2
MS-GPC-8- 10-57
Fab
LG2
7.2
MS-GPC-8-27-41
Fab
LG2
7.2
MS-GPC-8-27-41
Fab
PRIESS
7.7
MS-GPC-8
lgG4
PRIESS
8.3
MS-GPC-8-27-41
lgG4
PRIESS
1.1 ±0.1
MS-GPC-8-10-57
lgG4
PRIESS
1.1 ±0.2
MS-GPC-8-27-41
lgG4
LG2
1.23 ± 0.2
MS-GPC-8-10-57
lgG4
LG2
1.0 ±0.1
8D1
mlgG
PRIESS
33
L243
mlgG
PRIESS
47
75
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Table 7
IC50 values for certain anti-HLA-DR antibody fragments of the invention in an assay to
determine IL-2 secretion after antigen-specific stimulation of T-Hyb 1 cells. IC50 for the
IgG forms (bivalent) are represented as molar concentrations, while in order to provide
easy comparison, IC50s for the Fab forms (monovalent) are expressed in terms of half
the concentration of the Fab to enable direct comparison to IgG forms.
Anti-HI A.nR
aiiuuuuy irctyinmiL
rui \ 1 1
IC50
(IgG/nM)
((Fab)/2/nM)
IVIclAII MUI 1 ]
II II IIUI LIU! 1^/0^
Mean
ot
mq riDr 1 a -in cv
Mo-oKO-o- IU-D/
igij
U.o1
U.U1
1 uu
IV/1Q PDP Q 97 a a
igo
Kj.eLo
r\ r\-7
U.UY
l UU
IVIo-orO-o-O- 1 0
igvj
0.42
0.06
■\ nn
I uu
ig<j
3.6
1.1
i nn
1 uu
IVIo-o rU-o-D
igij
6.7
2.0
■i nn
1 uu
MS-GPC-8
IgG
11.0
0.8
100
MS-GPC-8-6-2
Fab
4.7
1.9
100
MS-GPC-8-6-13
Fab
2.1
0.8
100
MS-GPC-8-6-19
Fab
5.3
0.2
100
MS-GPC-8-10-57
Fab
2.9
1.0
100
MS-GPC-8-6-27
Fab
3.0
1.2
100
MS-GPC-8-6-47
Fab
2.6
0.6
100
MS-GPC-8-27-7
Fab
5.9
2.2
100
MS-GPC-8-27-10
Fab
7.3
1.9
100
MS-GPC-8-27-41
Fab
3.6
0.7
100
MS-GPC-8-6
Fab
20
100
MS-GPC-8
Fab
110
100
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Claims
1. A composition including a polypeptide comprising an antibody-based antigen-binding
domain of human composition with binding specificity for an antigen expressed on
the surface of a human cell, wherein treating cells expressing said antigen with a
multivalent polypeptide having two or more of said antigen-binding domains causes
or leads to killing of said cells in a manner where neither cytotoxic entities nor
immunological mechanisms are needed for said killing.
2. A composition including a polypeptide comprising an antibody-based antigen-binding
domain which binds to human HLA DR with a K d of 1jaM or less, wherein treating
cells expressing HLA DR with a multivalent polypeptide having two or more of said
antigen-binding domains causes or leads to killing of said cells in a manner where
neither cytotoxic entities nor immunological mechanisms are needed for said killing.
3. A composition including a multivalent polypeptide comprising a plurality of antibody-
based antigen-binding domains of human composition which specifically bind to
human HLA DR, wherein treating cells expressing HLA DR with said multivalent
polypeptide causes or leads to killing of said cells in a manner where neither
cytotoxic entities nor immunological mechanisms are needed for said killing, wherein
said antigen-binding domains individually bind to human HLA DR with a Kd of 1p,M or
less.
4. A composition including a multivalent polypeptide comprising a plurality of antibody-
based antigen-binding domains of human composition which specifically bind to
human HLA DR, wherein treating cells expressing HLA DR with said multivalent
polypeptide causes or leads to killing of said cells in a manner where neither
cytotoxic entities nor immunological mechanisms are needed for said cell killing,
wherein said multivalent polypeptide has an EC 50 of 100 nM or less for killing
activated lymphoid cells.
5. A composition including a polypeptide comprising at least one antibody-based
antigen-binding domain that binds to human HLA DR with a K d of l\xM or less, said
antigen-binding domain being isolated by a method which includes isolation of VL
and VH domains of human composition from a recombinant antibody library by ability
to bind to at least one epitope of human HLA DR, wherein treating cells expressing
HLA DR with a multivalent polypeptide having two or more of said antigen binding
domains causes or leads to killing of said cells in a manner where neither cytotoxic
entities nor immunological mechanisms are needed for said killing.
81
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6. The composition of claim 5, wherein the method for isolating the antigen-binding
domain includes the further steps of:
a. generating a library of variants of at least one of the CDR1 , CDR2 and CDR3
sequences of one or both of the VL and VH domains, and
b. isolation of VL and VH domains from the library of variants by ability to bind to
human HLA DR with a K d of 1 uM or less.
7. The composition of any of claims 1-6, wherein the multivalent polypeptide has an
EC 50 for killing transformed cells at least 5-fold lower than the EC 50 for killing normal
cells.
8. The composition of any of claims 1-6, wherein the multivalent polypeptide has an
EC 5 o for killing activated cells at least 5-fold lower than the EC 50 for killing unactivated
cells.
9. The composition of any of claims 1-6, wherein the multivalent polypeptide has an
EC 50 of 50nM or less for killing transformed cells.
10. The composition of any of claims 1-6, wherein the multivalent polypeptide has an
EC 50 for killing lymphoid tumor ceils of 10nM or less.
11. The composition of any of claim 1-6 or 8, wherein the multivalent polypeptide kills
activated lymphoid cells.
12. The composition of claim 11, wherein said activated lymphoid cells are lymphoid
tumor cells representing a disease selected from B cell non-Hodgkin lymphoma, B
cell lymphoma, B cell acute lymphoid leukemia, Burkitt lymphoma, Hodgkin
lymphoma, hairy cell leukemia, acute myeloid leukemia, T cell lymphoma, T cell non-
Hodgkin lymphoma, chronic myeloid leukemia, chronic lymphoid leukemia, and
multiple myeloid leukemia.
13. The composition of claim 11, wherein said activated lymphoid cells are from a cell
line taken from the list of Priess, GRANTA-519, KARPAS-422, KARPAS-299, DOHH-
2, SR-786, MHH-CALL-4, MN-60, BJAB, RAJI, L-428, HDLM-2, HD-MY-Z, KM-H2,
L1236, BONNA-12, HC-1, NALM-1, L-363, EOL-1, LP-1, RPMI-8226, and MHH-
PREB-1 cell lines.
14. The composition of any of claims 1-6, wherein the multivalent polypeptide has an
EC 50 of 100nM or less for killing cells of at least one of lymphoid tumor cell lines
selected from the list of KARPAS-422, DOHH-2, SR-7, MHH-CALL-4, MN-60, HD-
MY-Z, NALM-1 and LP-1 .
WO 01/87337
PCT/US01/15625
15. The composition of any of claims 1-6, wherein the multivalent polypeptide has an
EC 50 of 50nM or less for killing cells from at least one lymphoid tumor cell line
selected from the list of KARPAS-422, DOHH-2, MN-60, NALM-1 and LP-1.
16. The composition of any of claims 1-6, wherein the multivalent polypeptide hasan EC 50
of 10nM or less for killing cells from at least one B cell lymphoblastoid cell line
selected from the list LG2 and Priess.
17. The composition of any of claims 1-6, wherein said cells are non-lymphoid cells that
express MHC class II molecules
18. The composition of any of claims 1-6, wherein said antigen-binding domain binds to
the 0-chain of HLA-DR.
19. The composition of claim 18, wherein said antigen-binding domain binds to the first
domain of the B-chain of HLA-DR.
20. The composition of any of claims 1-6, wherein said antigen-binding domain binds to
one or more HLA-DR types selected from the group consisting of DR1-0101, DR2-
15021, DR3-0301, DR4Dw4-0401, DR4Dw1 0-0402, DR4Dw1 4-0404, DR6-1302,
DR6-1401, DR8-8031, DR9-9012, DRw53-B4*0101 and DRw52-B3*0101.
21. The composition of claim 20, wherein said antigen-binding domain binds to at least 5
different of said HLA-DR types.
22. The composition of any one of claims 1-6, wherein said antigen-binding domain
includes a combination of a VH domain and a VL domain, wherein said combination
is found in one of the clones taken from the list of MS-GPC-1, MS-GPC-6, MS-GPC-
8, MS-GPC-1 0, MS-GPC-8-1 , MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10, MS-GPC-
8-17, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-1 9, MS-GPC-8-6-
27, MS-GPC-8-6-45, MS-GPC-8-6-1 3, MS-GPC-8-6-47, MS-GPC-8-1 0-57, MS-GPC-
8-27-7, MS-GPC-8-27-10and MS-GPC-8-27-41.
23. The composition of any one of claims 1-6, wherein said antigen-binding domain
includes of a combination of HuCAL VH2 and HuCAL VA1, wherein the VH CDR3, VL
CDR1 And VL CDR3 is found, in one of the clones taken from the list of MS-GPC-1 ,
MS-GPC-8, MS-GPC-1 0, MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10,
MS-GPC-8-1 7, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-1 9, MS-
GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-1 3, MS-GPC-8-6-47, MS-GPC-8-1 0-57,
MS-GPC-8-27-7, MS-GPC-8-27-1 0 and MS-GPC-8-27-41.
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24. The composition of any one of claims 1-23, wherein said antigen-binding domain
includes a combination of HuCAL VH2 and HuCAL VA1, wherein the VH CDR3
sequence is taken from the consensus CDR3 sequence
nnnnRGnFDn
wherein each n independently represents any amino acid residue; and/or
wherein the VL CDR3 sequence is taken from the consensus CDR3 sequence
QSYDnnnn
wherein each n independently represents any amino acid residue.
25. The composition of claim 24, wherein the VH CDR3 sequence is SPRYGAFDY
and/or the VL CDR3 sequence is QSYDLIRH or QSYDMNVH.
26. The composition of any one of claims 1-23, wherein said antigen-binding domain
competes for antigen binding with an antibody including a combination of HuCAL
VH2 and HuCAL VA1, wherein the VH CDR3 sequence is taken from the consensus
CDR3 sequence
nnnnRGnFDn
each n independently represents any amino acid residue; and/or
the VL CDR3 sequence is taken from the consensus CDR3 sequence
QSYDnnnn
each n independently represents any amino acid residue.
27. The composition of claim 26, wherein the VH CDR3 sequence is SPRYGAFDY
and/or the VL CDR3 sequence is QSYDLIRH or QSYDMNVH.
28. The composition of any one of claims 1-27, wherein said antigen-binding domain
includes a VL CDR1 sequence represented in the general formula
SGSnnNIGnNYVn
wherein each n independently represents any amino acid residue.
29. The composition of claim 28, wherein the CDR1 sequence is SGSESNIGNNYVQ.
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30. The composition of any of claims 1-29, wherein the mechanism of said killing
involves an innate pre-programmed process of said cell.
31. The composition of claim 30, wherein said killing is non-apoptotic.
32. The composition of claim 30, wherein said killing is dependent on the action of non-
caspase proteases, and/or wherein said killing cannot be inhibited by zVAD-fmk or
zDEVD-fmk.
33. The composition of any one of claims 1-32, wherein said antibody-based antigen-
binding domain is part of a multivalent polypeptide including at least a F(ab') 2
antibody fragment or a mini-antibody fragment.
34. The composition of any one of claims 1-32, wherein said antibody-based antigen-
binding domain is part of a multivalent polypeptide comprising at least two
monovalent antibody fragments selected from Fv, scFv, dsFv and Fab fragments,
and further comprises a cross-linking moiety or moieties.
35. The composition of any one of claims 1-32, wherein said antibody-based antigen-
binding domain is part of a multivalent polypeptide comprising at least one full
antibody selected from the antibodies of classes lgG1 , 2a, 2b, 3, 4, IgA, and IgM.
36. The composition of any one of claims 1-32, wherein said antibody-based antigen-
binding domain is part of a multivalent polypeptide that is formed prior to binding to a
cell.
37. The composition of any one of claims 1-32, wherein said antibody-based antigen-
binding domain is part of a multivalent polypeptide that is formed after binding to a
cell.
38. The composition of claim 3 or 4, wherein the antigen binding sites are cross-linked to
a polymer.
39. A nucleic acid comprising a protein coding sequence for an antigen-binding domain
comprised in any of claims 1-32, or a multivalent polypeptide thereof.
40. A vector comprising the nucleic acid of claim 39, and a transcriptional regulatory
sequence operably linked thereto.
41 . A host cell harboring at least one nucleic acid of claim 39 or the vector of claim 40.
42. A method for the production of composition comprising a multivalent polypeptide that
causes or leads to killing of cells in a manner where neither cytotoxic entities nor
immunological mechanisms are needed for said killing, comprising culturing the cells
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of claim 41 under conditions wherein the nucleic acid is expressed either as a
multivalent polypeptide or as a polypeptide comprising at least one antigen binding
domains which is subsequently treated to form a multivalent polypeptide composition.
43. The composition of any of claims 1 -38, formulated in a pharmaceutical^ acceptable
carrier and/or diluent.
44. The use of a composition of any of claims 1-38, for preparing a pharmaceutical
preparation for the treatment of animals.
45. The use of a nucleic acid of claim 39 for preparing a pharmaceutical preparation for
the treatment of animals
46. The use of a host cell of claim 41 for preparing a pharmaceutical preparation for the
treatment of animals
47. The use of the method of claim 42 for preparing a pharmaceutical preparation for the
treatment of animals
48. The use according to claim 44-47, wherein said animal is a human.
49. The use according to claim 44-48, for the treatment of cell proliferative disorders,
wherein said antibody-based antigen binding domain is part of a multivalent
polypeptide.
50. The use according to claim 49, wherein said treatment is the treatment of disorders
involving transformed cells expressing MHC class II antigens.
51. The use according claim 49 or 50, wherein said treatment is the treatment of a
disorder selected from B cell non-Hodgkin lymphoma, B cell lymphoma, B cell acute
lymphoid leukemia, Burkitt lymphoma, Hodgkin lymphoma, hairy cell leukemia, acute
myeloid leukemia, T cell lymphoma, T cell non-Hodgkin lymphoma, chronic myeloid
leukemia, chronic lymphoid leukemia, and multiple myeloid leukemia.
52. The use according to any of claims 44-48, wherein said treatment is the treatment of
disorders involving unwanted activation of cells of the immune system, such as
lymphoid cells expressing MHC class II.
53. The use according to any of claims 44-48, wherein said treatment is the treatment of
a disorder selected from rheumatoid arthritis, juvenile arthritis, multiple sclerosis,
Grave's disease, insulin-dependent diabetes, narcolepsy, psoriasis, systemic lupus
erythematosus, ankylosing spondylitis, transplant rejection, graft vs. host disease,
Hashimoto's disease, myasthenia gravis, pemphigus vulgaris, glomerulonephritis,
thyroiditis, pancreatitis, insulitis, primary biliary cirrhosis, irritable bowel disease and
Sjogren syndrome.
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54. The use according to any of claims 44-48, wherein said disorder is selected from
myasthenia gravis, rheumatoid arthritis, multiple sclerosis, transplant rejection and
graft vs. host disease.
55. A diagnostic composition including the composition of any of claims 1-38.
56. A diagnostic composition including the composition of any of claims 1-38 and a
cross-linking moiety or moieties.
57. A method for killing a cell expressing an antigen on the surface of said cell
comprising the step of treating the cell with a plurality of antigen-binding domains of
any one of claims 1-38, wherein said antibody-based antigen-binding domains are
part of a multivalent polypeptide, and where neither cytotoxic entities nor
immunological mechanisms are needed to causes or leads to said killing
58. A method to identify patients that can be treated with a composition of any of claims
1-38, formulated in a pharmaceutically acceptable carrier and/or diluent comprising
the steps of
a. Isolating cells from a patient;
, b. Contacting said cells with the composition of any of claims 1-38; and
c. Measuring the degree of killing or immunosuppression of said cells.
59. A kit to identify patients that can be treated with a composition of any of claims 1-38,
formulated in a pharmaceutically acceptable carrier and/or diluent comprising
a. A composition of any of claims 1-38; and
b. Means to measure the degree of killing or immunosuppression of said cells.
60. A kit comprising
a. a composition according to any one of claims 1-38, and
b. a cross-linking moiety.
61 . A kit comprising
a. a composition according to any one of claims 1-38, and
b. a detectable moiety or moieties, and
c. reagents and/or solutions to effect and/or detect binding of (i) to an antigen.
62. A cytotoxic composition comprising a composition of any one of claims 1-38 operably
linked to a cytotoxic agent.
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63. An immunogenic composition comprising a composition of any one of claims 1-38
operably linked to an immunogenic agent.
64. A method to kill a cell comprising contacting said cell with a composition of any one
of claims 1-38 operably linked a cytotoxic or immunogenic agent.
65. The use of a composition of any one of claims 1-38 operable linked a cytotoxic or
immunogenic agent for preparing a pharmaceutical preparation for the treatment of
animals.
66. A composition including a polypeptide comprising at least one antibody-based
antigen-binding domain with a binding specificity for a human MHC class II antigen
with a K d of 1uM or less, wherein treating cells expressing said antigen with said
polypeptide causes or leads to suppression of an immune response.
67. A composition including a polypeptide comprising at least one antibody-based
antigen-binding domain with a binding specificity for human HLA DR antigen,
wherein treating cells expressing HLA DR with said polypeptide causes or leads to
suppression of an immune response, and wherein said antigen-binding domain
includes a combination of a VH domain and a VL domain, wherein said
combination is found in one of the clones taken from the list of MS-GPC-1, MS-
GPC-6, MS-GPC-8, MS-GPC-1 0, MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-
GPC-8-10, MS-GPC-8-1 7, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-
GPC-8-6-19, MS-GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-13, MS-GPC-8-6-
47, MS-GPC-8-1 0-57, MS-GPC-8-27-7, MS-GPC-8-27-10 and MS-GPC-8-27-41.
68. A composition including a polypeptide comprising at least one antibody-based
antigen-binding domain with a binding specificity for a human MHC class II antigen
with a K d of 1uM or less, said antigen-binding domain being isolated by a method
which includes isolation of VL and VH domains of human composition from a
recombinant antibody library by ability to bind to human MHC class II antigen,
wherein treating cells expressing MHC Class II with said polypeptide causes or leads
to suppression of an immune response.
69. The composition of claim 68, wherein the method for isolating the antigen-binding
domain includes the further steps of:
a. generating a library of variants at least one of the CDR1, CDR2 and CDR3
sequences of one or both of the VL and VH domains, and
b. isolation of VL and VH domains from the library of variants by ability to bind to
human MHC class II antigen with a K d of 1uM or less;
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c. (optionally) repeating steps (a) and (b) with at least one other of the CDR1 ,
CDR2 and CDR3 sequences.
70. The composition of any of claims 67, 68 or 69, wherein said antigen-binding
domain binds to HLA-DR
71 . The composition of any of claims 66 or 70 wherein said antigen-binding domain binds
to the p-chain of HLA-DR.
72. The composition of claim 71, wherein said antigen-binding domain binds to an
epitope of the first domain of the p-chain of HLA-DR.
73. The composition of any of claims 66-72, wherein said cells are lymphoids cells.
74. The composition of any of claims 66-72, wherein said cells are non-lymphoid cells
and express MHC class II antigens.
75. The composition of any of claims 66-74, having an IC 50 for suppressing an immune
response of 1 u M or less.
76. The composition of any of claims 66-74, having an IC50 for inhibition of IL-2
secretion of 1 u M or less
77. The composition of any of claims 66-74, having an IC50 for inhibiting T cell
proliferation of 1 u M or less
78. The composition of any of claims 66-77, wherein said antigen-binding domain binds
to one or more HLA-DR types selected from the group consisting of DR1-0101, DR2-
15021, DR3-0301, DR4Dw4-0401, DR4Dw10-0402, DR4Dw14-0404, DR6-1302,
DR6-1401, DR8-8031, DR9-9012, DRw53-B4*0101 and DRw52-B3*0101.
79. The composition of claim 78, wherein said antigen-binding domain binds to at least 5
different of said HLA-DR types.
80. The composition of any of claims 66-79, wherein said antigen-binding domain
includes a combination of a VH domain and a VL domain, wherein said combination
is found in one of the clones taken from the list of MS-GPC-1 , MS-GPC-6, MS-
GPC-8, MS-GPC-1 0, MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-1 0,
MS-GPC-8-17, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-19,
MS-GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-1 3, MS-GPC-8-6-47, MS-GPC-
8-1 0-57, MS-GPC-8-27-7, MS-GPC-8-27-10 and MS-GPC-8-27-41 .
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81. The composition of any one of claims 66-77, wherein said antigen-binding domain
includes of a combination of HuCAL VH2 and HuCAL VA1 , wherein the VH CDR3, VL
CDR1 And VL CDR3 is found in one of the clones taken from the list of MS-GPC-1,
MS-GPC-8, MS-GPC-1 0, MS-GPC-8-1, MS-GPC-8-6, MS-GPC-8-9, MS-GPC-8-10,
MS-GPC-8-17, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-GPC-8-6-19, MS-
GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-13, MS-GPC-8-6-47, MS-GPC-8-1 0-57,
MS-GPC-8-27-7, MS-GPC-8-27-10 and MS-GPC-8-27-41 .
82. The composition of any of claims 66-77, wherein said antigen-binding domain
includes a combination of HuCAL VH2 and HuCAL VA1, wherein the VH CDR3
sequence is taken from the consensus CDR3 sequence
nnnnRGnFDn
wherein each n independently represents any amino acid residue; and/or
wherein the VL CDR3 sequence is taken from the consensus CDR3 sequence
QSYDnnnn
wherein each n independently represents any amino acid residue.
83. The composition of claim 82, wherein the VH CDR3 sequence is SPRYGAFDY
and/or the VL CDR3 sequence is QSYDLIRH or QSYDMNVH.
84. The composition of any of claims 66-77, wherein said antigen-binding domain
competes for antigen binding with an antibody including a combination of HuCAL
VH2 and HuCAL VA1, wherein the VH CDR3 sequence is taken from the consensus
CDR3 sequence
nnnnRGnFDn
each n independently represents any amino acid residue; and/or
the VL CDR3 sequence is taken from the consensus CDR3 sequence
QSYDnnnn
each n independently represents any amino acid residue.
85. The composition of claim 84, wherein the VH CDR3 sequence is SPRYGAFDY
and/or the VL CDR3 sequence is QSYDLIRH or QSYDMNVH.
86. The composition of any of claims 66-85, wherein said antigen-binding domain
includes a VL CDR1 sequence represented in the general formula
SGSnnNIGnNYVn
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wherein each n independently represents any amino acid residue.
87. The composition of claim 86, wherein the CDR1 sequence is SGSESNiGNNYVQ.
88. The composition of any one of claims 66-85, wherein said suppression of an immune
response is brought about by or manifests itself in down-regulation of expression of
said antigen expressed on the surface of said cell.
89. The composition of any one of claims 66-85, wherein said suppression of an immune
response is brought about by or manifests itself in inhibition of the interaction
between said cell and other cells, wherein said interaction would normally lead to an
immune response.
90. The composition of any one of claims 66-85, wherein said suppression of the
immune response is brought about by or manifests itself in the killing of said cells.
91 . The composition of claim 90, wherein said killing is mediated by binding of a plurality
of antigen-binding domains, wherein said antibody-based antigen-binding domains
are part of a multivalent polypeptide, and where neither cytotoxic entities nor
immunological mechanisms are needed to causes or leads to said killing.
92. The composition of any one of claims 66-91, formulated in a pharmaceutically
acceptable carrier and/or diluent
93. A pharmaceutical preparation comprising the composition of claim 75 in an amount
sufficient to suppress an immune response in an animal.
94. A pharmaceutical preparation comprising the composition of claim 76 in an amount
sufficient to inhibit IL-2 secretion in an animal.
95. A pharmaceutical preparation comprising the composition of claim 77 in an amount
sufficient to inhibit T cell proliferation in an animal.
96. The use of a composition of any one of claims 66-91, for preparing a pharmaceutical
preparation for the treatment of animals, such as where said animals are human.
97. A nucleic acid including a protein coding sequence for a polypeptide of the
composition of any of claims 66-91 .
98. A vector comprising the coding sequence of claim 97, and a transcriptional regulatory
sequence operably linked thereto.
99. A host cell harboring a nucleic acid of claim 97 or the vector of claim 98.
100. A method for the production of an immunosuppressive composition, comprising
culturing the cells of claim 99 under conditions wherein the nucleic acid is expressed.
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101. A method for suppressing activation of a cell of the immune system, such as
expressing HLA DR, comprising treating the cell with a composition of any of claims
66-92.
102. A method for suppressing proliferation of a cell of the immune system, such as
expressing HLA DR, comprising treating the cell with a composition of any of claims
66-92.
103. A method for suppressing IL-2 secretion by a cell of the immune system, such as
expressing HLA DR, comprising treating the cell with a composition of any of claims
66-92
104. A method for immunosuppressing a patient, comprising administering to the patient
an effective amount of a composition of any of claims 66-92 to reduce the level of
immunological responsiveness in the patient.
105. A method for killing a cell expressing an antigen on the surface of said cell
comprising the step of treating the cell with a plurality of antigen-binding domains of
any one of claims 66-87, wherein said antibody-based antigen-binding domains are
part of a multivalent polypeptide, and where neither cytotoxic entities nor
immunological mechanisms are needed to causes or leads to said killing, such where
said antigen is HLA DR.
106. The use according to claim 96, wherein said treatment is the treatment of a disorder
selected from rheumatoid arthritis, juvenile arthritis, multiple sclerosis, Grave's
disease, insulin-dependent diabetes, narcolepsy, psoriasis, systemic lupus
erythematosus, ankylosing spondylitis, transplant rejection, graft vs. host disease,
Hashimoto's disease, myasthenia gravis, pemphigus vulgaris, glomerulonephritis,
thyroiditis, pancreatitis, insulitis, primary biliary cirrhosis, irritable bowel disease and
Sjogren syndrome.
107. The use according to claim 96, wherein said treatment is the treatment of a disorder
selected from myasthenia gravis, rheumatoid arthritis, multiple sclerosis, transplant
rejection and graft vs. host disease.
108 A method of suppressing the interaction of a cell of the immune system with an other
cell, comprising contacting the cell with the composition of any of claims 66-92.
109. A method for conducting a pharmaceutical business comprising:
(i) isolating one or more antigen-binding domains that bind to antigens
expressed on the surface of human cells;
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(ii) generating a multivalent composition, such as multivalent polypeptide,
comprising a plurality of said antigen-binding domains, which multivalent
composition kills with an EC 50 of 50nM or less transformed or activated cells
that express said antigen, where neither cytotoxic entities nor immunological
mechanisms are needed to cause or lead to said killing.;
(iii) conducting therapeutic profiling of the multivalent composition, for efficacy
and toxicity in animals;
(iv) preparing a package insert describing the multivalent composition for
treatment of proliferative disorders; and
(v) marketing the multivalent composition for treatment of proliferative disorders.
110. A method for conducting a life science business comprising:
(i) isolating one or more antigen-binding domains that bind to antigens
expressed on the surface of human cells;
(ii) generating a multivalent composition, such as multivalent polypeptide,
comprising a plurality of said antigen-binding domains, which multivalent
composition kills with an EC 5 o of 50nM or less transformed or activated cells
expressing said antigen where neither cytotoxic entities nor immunological
mechanisms are needed to cause or lead to said killing;
(iii) licensing, jointly developing or selling, to a third party, the rights for selling the
multivalent composition.
111. The method of any of claims 109 or 110, wherein the antigen-binding domain, is
isolated by a method which includes
a. isolation of VL and VH domains of human composition from a recombinant
antibody library by ability to bind to HLA DR,
b. generating a library of variants at least one of the CDR1, CDR2 and CDR3
sequences of one or both of the VL and VH domains, and
c. isolation of VL and VH domains from the library of variants by ability bind to
HLA DR with a K d of 1 uM or less.
112. A method for conducting a pharmaceutical business comprising:
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(i) isolating one or more antigen-binding domains that bind to MHC class II
expressed on the surface of human cells with a K d of 1uM or less;
(ii) generating a composition comprising said antigen-binding domains, which
composition is immunosuppressant with an IC 5 o of 100nM or less;
(iii) conducting therapeutic profiling of the composition for efficacy and toxicity in
animals;
(iv) preparing a package insert describing the use of the composition for
immunosuppression therapy; and
(v) marketing the composition for use as an immunosuppressant.
113. A method for conducting a life science business comprising:
(i) isolating one or more antigen-binding domains that bind to MHC class II
expressed on the surface of human cells with a K d of 1uM or less;
(ii) generating a composition comprising said antigen-binding domains, which
composition is immunosuppressant with an IC 50 of 100nM or less;
(iii) licensing, jointly developing or selling, to a third party, the rights for selling the
composition.
114. The method of any of claims 112 or 113, wherein the antigen-binding domain is
isolated by a method which includes
a. isolation of VL and VH domains of human composition from a recombinant
antibody library by ability to bind to HLA DR,
b. generating a library of variants at least one of the CDR1 , CDR2 and CDR3
sequences of one or both of the VL and VH domains, and
c. isolation of VL and VH domains from the library of variants by ability to bind to
HLA DR with a Kd of 1 uM or less.
115. The method of any of claims 109-114, wherein said antigen-binding domain
comprises a combination of VH and VL domains found in the clones taken from the
list of MS-GPC-1, MS-GPC-8, MS-GPC-10, MS-GPC-8-1 , MS-GPC-8-6, MS-GPC-8-
9, MS-GPC-8-1 0, MS-GPC-8-1 7, MS-GPC-8-1 8, MS-GPC-8-27, MS-GPC-8-6-2, MS-
GPC-8-6-19, MS-GPC-8-6-27, MS-GPC-8-6-45, MS-GPC-8-6-13, MS-GPC-8-6-47,
MS-GPC-8-1 0-57, MS-GPC-8-27-7, MS-GPC-8-27-10 and MS-GPC-8-27-41.
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MS-GPC-
8-6
-0,018
-0,019
-0,012
-0,072
-0,016
1,306
MS-GPC-
8
-0,022
-0,016
-0,009
-0,081
-0,014
1,058
MS-GPC-
8-6-27
0,007
0,003
0,002
0,006
-0,004
1,297
MS-GPC-
8-10-57
0,005
0,003
0,003
0,014
-0,008
1,256
MS-GPC-
8-6-47
-0,001
0,008
0,011
0,013
-0,005
MS-GPC-
8-27-41
-0,025
-0,022
-0,007
-0,073
-0,018
1,525
MS-GPC-
8-6-13
-0,022
-0,021
-0,012
-0,079
-0,018
1,467
MS-GPC-
8-27-10
-0,02
-0,019
-0,01
-0,079
-0,016
1,493
MS-GPC-
8-27-7
-0,004
-0,003
-0,005
-0,005
-0,009
1,549
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WO 01/87337
PCT/US01/15625
WO 01/87337
PCT/US01/15625
9/49
Figure 7b
WO 01/87337
PCT/US01/15625
10/49
Figure 7c
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PCT/US01/15625
11/49
Figure 7d
WO 01/87337
PCT/US01/15625
12/49
Figure 8a
Mechanism of cell death
100
0 2 4 6
Incubation time (h)
WO 01/87337
PCT/US01/15625
13/49
Figure 8b
Mechanism of cell death
WO 01/87337
PCT/US01/15625
14/49
Figure 9a
WO 01/87337
PCT/US01/15625
15/49
Figure 9b
WO 01/87337
PCT/US01/15625
WO 01/87337
PCT/US01/15625
17/49
Figure 11
M13Reverse primer (100.0%)
SDSl
lac operator
mrnalacoperon\ \
-35
CAP SITE"
-35 cat
Bsp EI (173)
/ /CDR1
l I 1 I FW2
Sill! j
////// / X! '° r (233)
, ' ' / // j j Am I (233)
ll///f
-35
T->A
ColEI Bd2orign
Apa LI (2203)
VH1A
Bsl EII (301)
£ag 1(372)
■Bis Iffl (387)
SC I (430)
Ce! II (454)
Blp I (454)
LINKER
£co RV (520)
Sex AI (556)
f&Acc 651(618)
Xpn 1(622)
"ma I (632)
va 1(632)
I (634)
fisu 361(679)
|Myc-tag
Arte
glllseq9(1CX).0%)
Cla I (1035)
ipid HI (1362)
'OQII3(100.0%)
Genll-Nick
fl crig'n
WO 01/87337 PCT/US01/15625
18/49
Figure 11 (cont)
XbalSphI
1 AGAGCATGCG TAGGAGAAAA TAAAATGAAA CAAAGCACTA TTGCACTGGC
TCTCGTACGC ATCCTCTTTT ATTTTACTTT GTTTCGTGAT AACGTGACCG
51 ACTCTTACCG TTGCTCTTCA CCCCTGTTAC CAAAGCCGAC TACAAA.GATG
TGAGAATGGC AACGAGAAGT GGGGACAATG GTTTCGGCTG ATGTTTCTAC
Mf el
101 AAGTGCAATT GGTTCAGTCT GGCGCGGAAG TGAAAAAACC GGGCAGCAGC
TTCACGTTAA CCAAGTCAGA CCGCGCCTTC ACTTTTTTGG CCCGTCGTCG
BspEI
151 GTGAAAGTGA GCTGCAAAGC CTCCGGAGGC ACTTTTAGCA GCTATGCGAT
. CACTTTCACT CGACGTTTCG GAGGCCTCCG TGAAAATCGT CGATACGCTA
Xhol
Aval
2 01 TAGCTGGGTG CGCCAAGCCC CTGGGCAGGG TCTCGAGTGG ATGGGCGGCA
ATCGACCCAC GCGGTTCGGG GACCCGTCCC AGAGCTCACC TACCCGCCGT
BstEII
2 51 TTATTCCGAT TTTTGGCACG GCGAACTACG CGCAGAAGTT TCAGGGCCGG
AATAAGGCTA AAAACCGTGC CGCTTGATGC GCGTCTTCAA AGTCCCGGCC
BstEII
3 01 GTGACCATTA CCGCGGATGA AAGCACCAGC ACCGCGTATA TGGAACTGAG
CACTGGTAAT GGCGC CTACT TTCGTGGTCG TGGCGCATAT ACCTTGACTC
EagI BssHII
3 51 CAGCCTGCGT AGCGAAGATA CGGCCGTGTA TTATTGCGCG CGTTATTATG
GTCGGACGCA TCGCTTCTAT GCCGGCACAT AATAACGCGC GCAATAATAC
Sty I
4 01 ATCGTATGTA TAATATGGAT TATTGGGGCC AAGGCACCCT GGTGACGGTT
TAG CATAC AT ATTATACCTA ATAACCCCGG TTCCGTGGGA CCACTGCCAA
BlpI
Celll
4 51 AGCTCAGCGG GTGGCGGTTC TGGCGGCGGT GGGAGCGGTG GCGGTGGTTC
TCGAGTCGCC CACCGCCAAG ACCGCCGCCA CCCTCGCCAC CGCCACCAAG
EcoRV
5 01 TGGCGGTGGT GGTTCCGATA TCGAACTGAC CCAGCCGCCT TCAGTGAGCG
WO 01/87337 PCT/US01/15625
19/49
ACCGCCACCA CCAAGGCTAT AGCTTGACTG GGTCGGCGGA AGTCACTCGC
SexAI
551 TTGCACCAGG TCAGACCGCG CGTATCTCGT GTAGCGGCGA TGCGCTGGGC
AACGTGGTCC AGTCTGGCGC GCATAGAGCA CATCGCCGCT ACGCGACCCG
Xmal
Kpnl Smal
Acc65I Aval
6 01 GATAAATACG CGAGCTGGTA CCAGCAGAAA CCCGGGCAGG CGCCAGTTCT
CTATTTATGC GCTCGACCAT GGTCGTCTTT GGGCCCGTCC GCGGTCAAGA
BSU36I
651 GGTGAT TTAT GATGATTCTG ACCGTCCCTC AGGCATCCCG GAACGCTTTA
C C AC TAAATA CTACTAAGAC TGGCAGGGAG TCCGTAGGGC CTTGCGAAAT
BamHI
701 GCGGATCCAA CAGCGGCAAC ACCGCGACCC TGACCATTAG CGGCACTCAG
CGCCTAGGTT GTCGCCGTTG TGGCGCTGGG ACTGGTAATC GCCGTGAGTC
BpuAI
Bbsl
751 GCGGAAGACG AAGCGGATTA TTATTGCCAG AGCTATGACG CT CATATGCG
CGCCTTCTGC TTCGCCTAAT AATAACGGTC TCGATACTGC GAGTATACGC
Hpal MscI EcoRI
8 01 TCCTGTGTTT GGCGGCGGCA CGAAGTTAAC CGTTCTTGGC CAGGAATTCG
AGGACACAAA CCGCCGCCGT GCTTCAATTG GCAAGAACCG GTCCTTAAGC
851 AGCAGAAGCT GATCTCTGAG GAGGAT CTGA ACTAGGGTGG TGGCTCTGGT
TCGTCTTCGA CTAGAGACTC CTCCTAGACT TGATCCCACC ACCGAGACCA
901 TCCGGTGATT TTGATTATGA AAAGATGGCA AACGCTAATA AGGGGGCTAT
AGGCCACTAA AAC TAATACT TTTCTACCGT TTGCGATTAT TCCCCCGATA
glllseq9 100.0%
951 GAC CGAAAAT GCCGATGAAA ACGCGCTACA GTCTGACGCT AAAGGCAAAC
CTGGCTTTTA CGGCTACTTT TGCGCGATGT CAGACTGCGA TTTCCGTTTG
Clal
10 01 TTGATTCTGT CGCTACTGAT TACGGTGCTG CTATCGATGG TTT CATTGGT
AACTAAGACA GCGATGACTA ATGCCACGAC GATAGCTACC AAAGTAAC C A
1051 GACGTTTCCG GCCTTGCTAA TGGTAATGGT GCTACTGGTG ATTTTGCTGG
CTGCAAAGGC CGGAACGATT ACCATTACCA CGATGAC CAC TAAAACGACC
WO 01/87337
PCT/US01/15625
20/49
1101 CTCTAATTCC CAAATGGCTC AAGTCGGTGA CGGTGATAAT TCACCTTTAA
GAGATTAAGG GTTTAC CGAG TTCAGCCACT GCCACTATTA AGTGGAAATT
1151 TGAATAATTT CCGTCAATAT TTACCTTCCC TCCCTCAATC GGTTGAATGT
ACTTATTAAA GGCAGT TATA AATGGAAGGG AGGGAGT TAG CCAACTTACA
12 01 CGCCCTTTTG TCTTTGGCGC TGGTAAACCA TATGAATTTT CTATTGATTG
GCGGGAAAAC AGAAAC CGCG ACCATTTGGT ATACTTAAAA GATAACTAAC
1251 TGACAAAATA AACTTATTCC GTGGTGTCTT TGCGTTTCTT TTATATGTTG
ACTGTTTTAT TTGAATAAGG C AC CACAGAA ACGCAAAGAA AATATACAAC
13 01 CCACCTTTAT GTATGTATTT TCTACGTTTG CTAACATACT GCGTAATAAG
GGTGGAAATA CATACATAAA AGATGCAAAC GATTGTATGA CGCATTATTC
Hindi I I
13 51 GAGTCTTGAT AAGCTTGACC TGTGAAGTGA AAAATGGCGC AGATTGTGCG
CTCAGAACTA TTCGAACTGG ACACTTCACT TTTTACCGCG TCTAACACGC
OGIII3 100.0%
14 01 ACATTTTTTT TGTCTGCCGT TTAATGAAAT TGTAAACGTT AATATTTTGT
TGTAAAAAAA ACAGACGGCA AATTACTTTA ACATTT GCAA TTATAAAACA
1451 TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT TAAC CAATAG
ATTTTAAGCG CAATTTAAAA ACAATTTAGT CGAGTAAAAA ATTGGTTATC
15 01 GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG
CGGCTTTAGC CGTTTTAGGG AATATTTAGT TTTCTTATCT GGCTCTATCC
1551 GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG
CAACT CACAA CAAGGT C AAA CCTTGTTCTC AGGTGATAAT TTCTTGCACC
16 01 ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA
TGAGGTTGCA GTTTCCCGCT TTTTGGCAGA TAGTCCCGCT ACCGGGTGAT
1651 CGAGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC
GCTCTTGGTA GTGGGATTAG TTCAAAAAAC CCCAGCTCCA CGGCATTTCG
1701 ACTAAA.TCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA
TGATTTAGCC TTGGGATTTC CCTCGGGGGC TAAATCTCGA ACTGCCCCTT
1751 AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAG CGAA AGGAGCGGGC
TCGGCCGCTT GCACCGCTCT TTCCTTCCCT TCTTTCGCTT TCCTCGCCCG
18 01 GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC
CGATCCCGCG ACCGTTCACA TCGCCAGTGC GACGCGCATT GGTGGTGTGG
1851 CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTGCTAGCCA TGTGAGCAAA
GCGGCGCGAA. TTACGCGGCG ATGTCCCGCG CACGATCGGT ACACTCGTTT
1901 AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT
TCCGGTCGTT TTCCGGTCCT TGGCATTTTT CCGGCGCAAC GAC CGCAAAA
1951 TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT
AGGTATCCGA GGCGGGGGGA CTGCTCGTAG TGTTTTTAGC TGCGAGTTCA
WO 01/87337
PCT/US01/15625
21/49
2 0 01 CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC
GTCTCCACCG CTTTGGGCTG TCCTGATATT TCTATGGTCC GCAAAGGGGG
2 051 TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT
ACCTTCGAGG GAGCACGCGA GAGGACAAGG CTGGGACGGC GAATGGCCTA
2101 ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA
TGGACAGGCG GAAAGAGGGA AGCCCTTCGC ACCGCGAAAG AGTATCGAGT
2151 CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG
GCGACATCCA TAGAGTCAAG CCACATCCAG CAAGCGAGGT TCGACCCGAC
ApaLI
22 01 TGTGCACGAA CCCCCCGTTC AGTCCGACCG CTGCGCCTTA TCCGGTAACT
ACACGTGCTT GGGGGGCAAG TCAGGCTGGC GACGCGGAAT AGGCCATTGA
2251 ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA
TAGCAGAACT CAGGTTGGGC CATTCTGTGC TGAATAGCGG TGACCGTCGT
23 01 GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA
CGGTGAC CAT TGTCCTAATC GTCTCGCTCC ATACATCCGC CACGATGTCT
23 51 GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGA ACAGTATTTG
CAAGAACTTC ACCACCGGAT TGATGC CGAT GTGATCTTCT TGTCATAAAC
24 01 GTATCTGCGC TCTGCTGTAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC
CATAGACGCG AGACGACATC GGTCAATGGA AGCCTTTTTC TCAACCATCG
2451 TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT TTTTTGTTTG
AGAACTAGGC CGTTTGTTTG GTGGCGACCA TCGCCACCAA AAAAACAAAC
2 5 01 CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA
GTTCGTCGTC TAATGCGCGT CTTTTTTTCC TAGAGTTCTT CTAGGAAACT
2551 TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG
AGAAAAGATG CCCCAGACTG CGAGTCACCT TGCTTTTGAG TGCAATTCCC
26 01 ATTTTGGTGA GAT CTAGCAC CAGGCGTTTA AGGGCACCAA TAACTGCCTT
TAAAAC CAGT CTAGATCGTG GTCCGCAAAT TCCCGTGGTT ATTGACGGAA
2651 AAAAAAATTA CGCCCCGCCC TGCCACTCAT CGCAGTACTG TTGTAATTCA
TTTTTTTAAT GGGGGGCGGG ACGGTGAGTA GCGTCATGAC AACATTAAGT
2 701 TTAAGCATTC TGCCGACATG GAAGCCATCA CAAACGGCAT GATGAACCTG
AATTCGTAAG ACGGCTGTAC CTTCGGTAGT GTTTGCCGTA CTACTTGGAC
2 751 AATCGCCAGC GGCATCAGCA CCTTGTCGCC TTGCGTATAA TATTTGCCCA
TTAGCGGTCG CCGTAGTCGT GGAACAG CGG AACGCATATT ATAAACGGGT
2 8 01 TAGTGAAAAC GGGGGCGAAG AAGTTGTCCA TATTGGCTAC GTTTAAATCA
ATCACTTTTG CCCCCGCTTC TTCAACAGGT ATAAC CGATG CAAATTTAGT
2 851 AAACTGGTGA AACTCACCCA GGGATTGGCT GAGACGAAAA ACATATTCTC
TTTGACCACT TTGAGTGGGT CCCTAACCGA CTCTGCTTTT TGTATAAGAG
WO 01/87337
PCT/US01/15625
22/49
2 901 AATAAACCCT TTAGGGAAAT AGGCCAGGTT TTCACCGTAA CACGCCACAT
TTATTTGGGA AATCCCTTTA TCCGGTCCAA AAGTGGCATT GTGCGGTGTA
2951 CTTGCGAATA TATGTGTAGA AACTGCCGGA AATCGTCGTG GTATTCACTC
GAACGCTTAT AT AC AC AT C T TTGACGGCCT TTAGCAGCAC CATAAGTGAG
3 0 01 CAGAGCGATG AAAACGTTTC AGTTTGCTCA TGGAAAACGG TGTAACAAGG
GTCTCGCTAC TTTTGCAAAG TCAAACGAGT ACCTTTTGCC ACATTGTTCC
3 051 GTGAACACTA TCCCATATCA CCAGCTCACC GTCTTTCATT GCCATACGGA
CACTTGTGAT AGGGTATAGT GGTCGAGTGG CAGAAAGTAA CGGTATGCCT
3101 ACTCCGGGTG AGCATTCATC AGGCGGGCAA GAATGTGAAT AAAGGCCGGA
TGAGGCCCAC TCGTAAGTAG TCCGCCCGTT CTTACACTTA TTTCCGGCCT
3151 TAAAACTTGT GCTTATTTTT CTTTACGGTC TTTAAAAAGG CCGTAATATC
ATTTTGAACA CGAATAAAAA GAAATGCCAG AAATTTTTCC GG CAT TAT AG
32 01 CAGCTGAACG GTCTGGTTAT AGGTACATTG AGCAACTGAC TGAAATGCCT
GTCGACTTGC CAGACCAA.TA TCCATGTAAC TCGTTGACTG ACTTTACGGA
3251 CAAAATGTTC TTTACGATGC CATTGGGATA TAT CAACGGT GGT AT AT C C A
GTTTTACAAG AAATGCTACG GTAACCCTAT ATAGTTGCCA CCATATAGGT
33 01 GTGATTTTTT TCTCCATTTT AGCTTCCTTA GCTCCTGAAA ATCTCGATAA
CACTAAAAAA AGAGGTAAAA TCGAAGGAAT CGAGGACTTT TAGAGCTATT
/
3 3 51 CTCAAAAAAT ACGCCCGGTA GTGATCTTAT TTCATTATGG TGAAAGTTGG
GAGTTTTTTA TGCGGGCCAT CACTAGAATA AAGTAATACC ACTTTCAACC
34 01 AACCTCACCC GACGTCTAAT GTGAGTTAGC TCACTCATTA GGCACCCCAG
TTGGAGTGGG CTGCAGATTA CACTCAATCG AGTGAGTAAT CCGTGGGGTC
3451 GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGGAA TTGTGAGCGG
CGAAATGTGA AATACGAAGG CCGAGCATAC AACACACCTT AACACTCGCC
M13 Reverse primer 100.0% Xbal
3501 ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GAATTTCT
TATTGTTAAA GTGTGTCCTT TGT CGATACT GGTACTAATG CTTAAAGA
WO 01/87337
PCT/US01/15625
23/49
Figure 12
i!
M13 Reverse primer (100.0%)
Iacpfo\\
MRNALAC OPERON \U
\ III
LAC OPERATOR \
.»>a I (2)
Sph I (12)
! SDSEQ
PHOA
!
FLAG
!
Mfe I (110)
FW1
i (i '
Hi
Bsp EI (176)
CDR1
i
/ FW2
jAva I £36)
'/
V.Xlio I (236)
III ll
CDR2
/VH3
A5jp V (326)
S/k 1 (326)
I i
\ III ii
I Hljj [fBstBI(12<S)
11/
RNAl^
-10
-35,
T->A
CdEI Ext2 origin
RNAII Primer
Apa LI (3068)"
replication start
Eag I (375)
Bss HE (390)
CDR3
SfJ> I (442)
Ce/ D (466)
I (466)
-35 lad
-10lacl
\ \\ ^iKdIH(939)
\ \\ s|'oaii3(ioao%)
i \l \\
i\ i ituncbortalsson
5 ll !
" Genll-Nick
n
T->G
G->C
recognition stemloop
Insert GGCCTTTTGCT
WO 01/87337 PCT/US01/15625
24/49
Figure 12 (cont)
Xbal SphI
1 TCTAGAGCAT GCGTAGGAGA AAATAAAATG AAACAAAGCA CTATTGCACT
AGATCTCGTA CGCATCCTCT TTTATTTTAC TTTGTTTCGT GATAACGTGA
51 GGCACTCTTA CCGTTGCTCT TCACCCCTGT TACCAAAGCC GACTACAAAG
CCGTGAGAAT GGCAACGAGA AGTGGGGACA ATGGTTTCGG CTGATGTTTC
Mf el
101 ATGAAGTGCA ATTGGTGGAA AGCGGCGGCG GCCTGGTGCA ACCGGGCGGC
TACTTCACGT TAACCACCTT TCGCCGCCGC CGGACCACGT TGGCCCGCCG
BspEI
151 AGCCTGCGTC TGAGCTGCGC GGCCTCCGGA TTTACCTTTA GCAGCTATGC
TCGGACGCAG ACTCGACGCG CCGGAGGCCT AAATGGAAAT CGTCGATACG
Xhol
Aval
2 01 GATGAGCTGG GTGCGCCAAG CCCCTGGGAA GGGTCTCGAG TGGGTGAGCG
CTACTCGACC CACGCGGTTC GGGGACCCTT CCCAGAGCTC ACCCACTCGC
251 CGATTAGCGG TAGCGGCGGC AGCAC CTATT ATGCGGATAG CGTGAAAGGC
GCTAATCGCC ATCGCCGCCG TCGTGGATAA TACGCCTATC GCACTTTCCG
BstBI
Sful
NspV
3 01 CGTTTTACCA TTTCACGTGA TAATT CGAAA AACACCCTGT ATCTGCAAAT
GCAAAATGGT AAAGTGCACT ATTAAGCTTT TTGTGGGACA TAGACGTTTA
EagI BssHII
351 GAACAGCCTG CGTGCGGAAG ATACGGCCGT GTATTATTGC GCGCGTGTTA
CTTGT CGGAC GCACGCCTTC TATGCCGGCA CATAATAACG CGCGCACAAT
Styl
4 01 AGAAGCATTT TTCTCGTAAG AATTGGTTTG ATTATTGGGG CCAAGGCACC
TCTTCGTAAA AAGAGCATTC TTAACCAAAC TAATAACCCC GGTTCCGTGG
WO 01/87337
PCT/US01/15625
25/49
Blpl
Celll
451 CTGGTGACGG TTAGCTCAGC GGGTGGCGGT TCTGGCGGCG GTGGGAGCGG
GACCACTGCC AATCGAGTCG CCCACCGCCA AGACCGCCGC CACCCTCGCC
EcoRV
5 01 TGGCGGTGGT TCTGGCGGTG GTGGTTCCGA TATCGTGATG ACCCAGAGCC
ACCGCCACCA AGACCGCCAC CACCAAGGCT ATAGCACTAC TGGGTCTCGG
PstI
551 CACTGAGCCT GCCAGTGACT CCGGGCGAGC CTGCGAGCAT TAGCTGCAGA
GTGACTCGGA CGGTCACTGA GGCCCGCTCG GACGCTCGTA ATCGACGTCT
Kpnl
Acc65I
6 01 AGCAGCCAAA GCCTGCTGCA TAGCAACGGC TATAACTATC TGGATTGGTA
TCGTCGGTTT CGGACGACGT ATCGTTGCCG ATATTGATAG ACCTAACCAT
Kpnl
Acc65I SexAI
651 CCTTCAAAAA CCAGGTCAAA GCCCGCAGCT ATTAATTTAT CTGGGCAGCA
GGAAGTTTTT GGTCCAGTTT CGGGCGTCGA TAATTAAATA GACCCGTCGT
BamHI
701 ACCGTGCCAG TGGGGTCCCG GATCGTTTTA GCGGCTCTGG ATCCGGCACC
TGGCACGGTC ACCCCAGGGC CTAGCAAAAT CGCCGAGACC TAGGCCGTGG
BpuAI
Bbsl
751 GATTTTACCC TGAAAATTAG CCGTGTGGAA GCTGAAGACG TGGGCGTGTA
CTAAAATGGG ACTTTTAATC GGCACACCTT CGACTTCTGC ACCCGCACAT
MscI
801 TTATTGCCAG CAGCATTATA CCACCCCGCC GACCTTTGGC CAGGGTACGA
AATAACGGTC GTCGTAATAT GGTGGGGCGG CTGGAAACCG GTCCCATGCT
WO 01/87337
PCT/US01/15625
26/49
BsiWI EcoRI
851 AAGTTGAAAT TAAACGTACG GAATT CGACT ATAAAGATGA CGATGACAAA
TTCAACTTTA ATTTGCATGC CTTAAGCTGA TATTTCTACT GCTACTGTTT
BssHII Hindlll
901 GGCGCGCCGT GGAGCCACCC GCAGTTTGAA AAATGATAAG CTTGACCTGT
CCGCGCGGCA CCTCGGTGGG CGTCAAACTT TTTACTATTC GAACTGGACA
OGIII3 100.0%
951 GAAGTGAAAA ATGGCGCAGA TTGTGCGACA TTTTTTTTGT CTGCCGTTTA
CTTCACTTTT TACCGCGTCT AACACGCTGT AAAAAAAACA GACGGCAAAT
OGIII3 100.0%
1001 AT T AAAGGGG GGGGGGGGCC GGC CTGGGGG GGGGTGTACA TGAAATTGTA
TAATTTCCCC CCCCCCCCGG CCGGACCCCC CCCCACATGT ACTTTAACAT
1051 AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC
TTG CAATTAT AAAACAATTT TAAGCGCAAT TTAAAAACAA TTTAGTCGAG
1101 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG
TAAAAAATTG GTTATCCGGC TTTAGC CGTT T T AGGGAAT A TTTAGTTTTC
1151 AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA C AAGAGT CCA
TTATCTGGCT CTATCCCAAC TCACAACAAG GTCAAACCTT GTTCTCAGGT
12 01 CTATTAAAGA ACGTGGACTC CAACGT CAAA GGGCGAAAAA CCGTCTATCA
GATAATTTCT TGCACCTGAG GTTGCAGTTT CCCGCTTTTT GGCAGATAGT
1251 GGGCGATGGC CCACTACGAG AACCATCACC CTAATCAAGT TTTTTGGGGT
CCCGCTACCG GGTGATGCTC TTGGTAGTGG GATTAGTTCA AAAAACCCCA
13 01 CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG CCCCCGATTT
GCTCCACGGC ATTTCGTGAT TTAGCCTTGG GATTTCCCTC GGGGGC TAAA
13 51 AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA
TCTCGAACTG CCCCTTTCGG CCGCTTGCAC CGCTCTTTCC TTCCCTTCTT
14 01 AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC
TCGCTTTCCT CGCCCGCGAT CCCGCGACCG TTCACATCGC CAGTGCGACG
1451 GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTGC
CGCATTGGTG GTGTGGGCGG CGCGAATTAC GCGGCGATGT CCCGCGCACG
WO 01/87337
PCT/US01/15625
27/49
15 01 TAGACTAGTG TTTAAACCGG AC CGGGGGGG GGCTTAAGTG GGCTGCAAAA
ATCTGATCAC AAATTTGGCC TGGCCCCCCC CCGAATTCAC CCGACGTTTT
1551 CAAAACGGCC TCCTGTCAGG AAGCCGCTTT TATCGGGTAG CCTCACTGCC
GTTTTGCCGG AGGACAGTCC TTCGGCGAAA ATAGCCCATC GGAGTGACGG
1601 CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATCAG TGAATCGGCC
GCGAAAGGTC AGCCCTTTGG ACAGCACGGT CGACGTAGTC ACTTAGCCGG
1651 AACGCGCGGG GAGAGG CGGT TTGCGTATTG GGAGCCAGGG TGGTTTTTCT
TTGCGCGCCC CTCTCCGCCA AACGCATAAC CCTCGGTCCC ACCAAAAAGA
1701 TTTCACCAGT GAGACGGGCA ACAGCTGATT GCCCTTCACC GCCTGGCCCT
AAAGTGGTCA CTCTGCCCGT TGTCGACTAA CGGGAAGTGG CGGACCGGGA
1751 GAGAGAGTTG CAGCAAGCGG TCCACGCTGG TTTGCCCCAG CAGGCGAAAA
CTCTCTCAAC GTCGTTCGCC AGGTGCGACC AAACGGGGTC GTCCGCTTTT
18 01 TCCTGTTTGA TGGTGGT CAG CGGCGGGATA TAACATGAGC TGTCCTCGGT
AGGACAAACT ACCACCAGTC GCCGCCCTAT ATTGTACTCG ACAGGAGCCA
1851 ATCGTCGTAT CCCACTACCG AGATGTCCGC ACCAACGCGC AGCCCGGACT
TAGCAGCATA GGGTGATGGC TCTACAGGCG TGGTTGCGCG TCGGGCCTGA
1901 CGGTAATGGC ACGCATTGCG CCCAGCGCCA TCTGATCGTT GGCAACCAGC
GCCATTACCG TGCGTAACGC GGGTCGCGGT AGACTAGCAA CCGTTGGTCG
1951 ATCGCAGTGG GAACGATGCC CTCATTCAGC ATTTGCATGG TTTGTTGAAA
TAGCGTCACC CTTGCTACGG GAGTAAGTCG TAAACGTACC AAACAACTTT
2001 ACCGGACATG GCACTCCAGT CGCCTTCCCG TTCCGCTATC GGCTGAATTT
TGGCCTGTAC CGTGAGGTCA GCGGAAGGGC AAGGCGATAG CCGACTTAAA
2 051 GATTGCGAGT GAGATATTTA TGCCAGCCAG CCAGACGCAG ACGCGCCGAG
CTAACGCTCA CTCTATAAAT ACGGTCGGTC GGTCTGCGTC TGCGCGGCTC
2101 ACAGAACTTA ATGGGCCAGC TAACAGCGCG ATTTGCTGGT GGCCCAATGC
TGTCTTGAAT TACCCGGTCG ATTGTCGCGC TAAACGACCA CCGGGTTACG
2151 GAC CAGATGC TCCACGCCCA GTCGCGTACC GTCCTCATGG GAGAAAATAA
CTGGTCTACG AGGTGCGGGT CAG CGCATGG CAGGAGTACC CTCTTTTATT
22 01 TACTGTTGAT GGGTGT CTGG T C AGAGAC AT CAAGAAATAA CGC CGGAACA
ATGACAACTA CCCACAGACC AGTCTCTGTA GTTCTTTATT GCGGCCTTGT
2251 TTAGTGCAGG CAGCTTCCAC AGCAATAGCA TCCTGGTCAT CCAGCGGATA
AATCACGTCC GTCGAAGGTG TCGTTATCGT AGGAC CAGTA GGTCGCCTAT
ApaLI
23 01 GTTAATAATC AGCCCACTGA CACGTTGCGC GAGAAGATTG TGCACCGCCG
CAATTATTAG TCGGGTGACT GTGCAACGCG CTCTTCTAAC ACGTGGCGGC
23 51 CTTTACAGGC TTCGACGCCG CTTCGTTCTA CCATCGACAC GACCACGCTG
GAAATGTCCG AAGCTGCGGC GAAGCAAGAT GGTAGCTGTG CTGGTGCGAC
WO 01/87337
PCT/US01/15625
28/49
24 01 GCACCCAGTT GATCGGCGCG AGATTTAATC GCCGCGACAA TTTGCGACGG
CGTGGGTCAA CTAGCCGCGC TCTAAATTAG CGGCGCTGTT AAACGCTGCC
2451 CGCGTGCAGG GCCAGACTGG AGGTGGCAAC GCCAATCAGC AACGACTGTT
GCGCACGTCC CGGTCTGACC TCCACCGTTG CGGTTAGTCG TTGCTGACAA
2501 TGCCCGCCAG TTGTTGTGCC ACGCGGTTAG GAATGTAAT T CAGCTCCGCC
ACGGGCGGTC AACAACACGG TGCGCCAATC CTTACATTAA GTCGAGGCGG
2 551 ATCGCCGCTT CCACTTTTTC CCGCGTTTTC GCAGAAACGT GGCTGGCCTG
TAGCGGCGAA GGTGAAAAAG GGCGCAAAAG CGTCTTTGCA CCGACCGGAC
2 601 GTTCACCACG CGGGAAACGG TCTGATAAGA GACACCGGCA TACTCTGCGA
CAAGTGGTGC GCCCTTTGCC AGACTATTCT CTGTGGCCGT ATGAGACGCT
2651 CAT CGTATAA CGTTACTGGT TTCACATTCA CCACCCTGAA TTGACTCTCT
GTAGCATATT GCAATGACCA AAGTGTAAGT GGTGGGACTT AACTGAGAGA
2701 TCCGGGCGCT ATCATGCCAT ACCGCGAAAG GTTTTGCGCC ATTCGATGCT
AGGCCCGCGA TAGTACGGTA TGGCGCTTTC CAAAACGCGG TAAGCTACGA
2751 AGCCATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG
TCGGTACACT CGTTTTCCGG TCGTTTTCCG GTCCTTGGCA TTTTTCCGGC
2801 CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA
GCAACGACCG CAAAAAGGTA TCCGAGGCGG GGGGACTGCT CGTAGTGTTT
2 851 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA
TTAGCTGCGA GTTCAGTCTC CACCGCTTTG GGCTGTCCTG ATATTTCTAT
2901 CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC
GGTCCGCAAA GGGGGACCTT CGAGGGAGCA CGCGAGAGGA CAAGGCTGGG
2 951 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG
ACGGCGAATG GCCTATGGAC AGGCGGAAAG AGGGAAGCCC TTCGCACCGC
3 001 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG
GAAAGAGTAT CGAGTGCGAC ATCCATAGAG TCAAGCCACA TCCAGCAAGC
ApaLI
3 051 CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG
GAGGTTCGAC CCGACACACG TGCTTGGGGG GCAAGTCGGG CTGGCGACGC
3101 CCTTATCCGG TAACTATCGT CTTGAGTCCA AC C CGGTAAG ACACGACTTA
GGAATAGGCC ATTGATAGCA GAACTCAGGT TGGGCCATTC TGTGCTGAAT
3151 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT
AGCGGTGACC GTCGTCGGTG ACCATTGTCC TAATCGTCTC GCTCCATACA
32 01 AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA
TCCGCCACGA TGTCTCAAGA ACTTCACCAC CGGATTGATG CCGATGTGAT
3251 GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGTAGCCAGT TACCTTCGGA
CTTCTTGTCA TAAAC CAT AG ACGCGAGACG ACATCGGTCA ATGGAAGCCT
WO 01/87337
PCT/US01/15625
29/49
33 01 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG
TTTTCTCAAC CATCGAGAAC TAGGCCGTTT GTTTGGTGGC GACCATCGCC
33 51 TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC
ACCAAAAAAA CAAACGTTCG TCGTCTAATG CGCGTCTTTT TTTC CTAGAG
34 01 AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA
TTCTTCTAGG AAACTAGAAA AGATGCCCCA GACTGCGAGT CACCTTGCTT
3451 AACTCACGTT AAGGGATTTT GGTCAGATCT AGCACCAGGC GTTTAAGGGC
TTGAGTGCAA TTCCCTAAAA CCAGTCTAGA TCGTGGTCCG CAAATTCCCG
3 5 01 AC CAATAACT GCCTTAAAAA AATTACGCCC CGCCCTGCCA CTCATCGCAG
TGGTTATTGA CGGAATTTTT TTAATGCGGG GCGGGACGGT GAGTAGCGTC
3 551 TACTGTTGTA AT T CAT T AAG CATTCTGCCG ACATGGAAGC CATCACAAAC
ATGACAACAT TAAGTAATTC GTAAGACGGC TGTACCTTCG GTAGTGTTTG
3 601 GGCATGATGA ACCTGAATCG CCAGCGGCAT CAGCACCTTG TCGCCTTGCG
CCGTACTACT TGGACTTAGC GGTCGCCGTA GTCGTGGAAC AGCGGAACGC
3 651 TATAATATTT GCCCATAGTG AAAACGGGGG CGAAGAAGTT GTCCATATTG
ATATTATAAA CGGGTATCAC TTTTGCCCCC GCTTCTTCAA CAGGTATAAC
3 701 GCTACGTTTA AAT CAAAACT GGTGAAACTC ACCCAGGGAT TGGCTGAGAC
CGATGCAAAT TTAGTTTTGA CCACTTTGAG TGGGTCCCTA ACCGACTCTG
3 751 GAAAAACATA TTCTCAATAA ACCCTTTAGG GAAATAGGCC AGGTTTTCAC
CTTTTTGTAT AAGAGTTATT TGGGAAATCC CTTTATCCGG TCCAAAAGTG
3801 CGTAACACGC CACATCTTGC GAAT AT AT GT GTAGAAACTG CCGGAAATCG
GCATTGTGCG GTGTAGAACG CTTATATACA CATCTTTGAC GGCCTTTAGC
3 851 TCGTGGTATT CACTCCAGAG CGATGAAAAC GTTTCAGTTT GCTCATGGAA
AG C AC C ATAA GTGAGGTCTC GCTACTTTTG C AAAGT CAAA CGAGTACCTT
3 901 AACGGTGTAA CAAGGGTGAA CACTATCCCA TAT CAC C AG C TCACCGTCTT
TTGCCACATT GTTCCCACTT GTGATAGGGT ATAGTGGTCG AGTGGCAGAA
3 951 T CATTGC CAT ACGGAACTCC GGGTGAGCAT TCATCAGGCG GGCAAGAATG
AGTAACGGTA TGCCTTGAGG CCCACTCGTA AGTAGTCCGC CCGTTCTTAC
4 0 01 TGAATAAAGG CCGGATAAAA CTTGTGCTTA TTTTTCTTTA CGGTCTTTAA
ACTTATTTCC GGCCTATTTT GAACACGAAT AAAAAGAAAT GCCAGAAATT
4 051 AAAGGC CGTA ATATCCAGCT GAACGGTCTG GTTATAGGTA CATTGAGCAA
TTTCCGGCAT TATAGGTCGA CTTGCCAGAC CAATATCCAT GTAACT CGTT
4101 CTGACTGAAA TGCCTCAAAA TGTTCTTTAC GATGCCATTG GGATATATCA
GACTGACTTT ACGGAGTTTT ACAAGAAATG CTACGGTAAC CCTATATAGT
4151 ACGGTGGTAT ATCCAGTGAT TTTTTTCTCC ATTTTAGCTT CCTTAGCTCC
TGCCACCATA TAGGTCACTA AAAAAAGAGG TAAAATCGAA GGAATCGAGG
42 01 TGAAAATCTC GATAACTCAA AAAATACGCC CGGTAGTGAT CTTATTTCAT
ACTTTTAGAG CTATTGAGTT TTTTATGCGG GCCATCACTA GAATAAAGTA
WO 01/87337 PCT/US01/15625
30/49
42 51 TATGGTGAAA GTTGGAACCT CACCCGACGT CTAATGTGAG TTAGCTCACT
ATACCACTTT CAACCTTGGA GTGGGCTGCA GATTACACTC AATCGAGTGA
43 01 CATTAGGCAC CCCAGGCTTT ACACTTTATG CTTCCGGCTC GTATGTTGTG
GTAATCCGTG GGGTCCGAAA TGTGAAATAC GAAGGCCGAG CATACAACAC
M13 Reverse primer 100.0%
43 51 TGGAATTGTG AGCGGATAAC AATTTCACAC AGGAAACAGC TATGACCATG
ACCTTAACAC TCGCCTATTG TTAAAGTGTG TCCTTTGTCG ATACTGGTAC
44 01 ATTACGAATT
TAATGCTTAA
WO 01/87337
PCT/US01/15625
31/49
Figure 13
cmpA- signal sequence
SDSeq I
Xba I (4935)j \
\\
M13Reversepnrrcr (100.0%) | j
SDSEfc) |!
MRNA LAC OPERON
LAC OPERATOR: \ (
I I
Eco RV(1)
Sex AI (37)
j Acc 651(105)
I I
! \Kpn 1(109)
deletionofT \ | \ 111
\ \\\\\
CAP 9TE\ \| ! 1 I
|| jjXma 1(119)
jjAva 1(119)
Msma 1(121)
til Bsu 361 (166)
I'll 1
Hi I Bamiil(191)
Bbs 1(249)
Bpu AI(249)
Bsu 361 (282)
Msc 1(325)
Z>ra in (345)
'CL lambda
'Stu I (636)
RNA1I Primer y
Apa LI (3591)
replication start
-35 lad
-10 lad
Apa LI (2864)
7;o 1(867)
Sva 1(867)
,Nsp V(954)
S> I (954)
1st BI (954)
to HO (1018)
[Sty I (1061)
'cy n (1085)
VBlp I (1085)
||CH1
>\\ ItoR I (1395)
1 '
",\ 'FLAG(M2)
HH (1426)
.(streptagll
tin dm (1462)
i j OSII3 (100.0%)
| ftrcticnal ssori
t
Genii-Nick
A H
I \ G->C
| reccgriticn sternlccp
Insert GGCCTTTTGCT
WO 01/87337
EcoRV
1 ATCGTGCTGA
TAGCACGACT
5 1 GACCATCTCG
CTGGTAGAGC
Kpnl
Acc65I
101 GCTGGTACCA
CGACCATGGT
151 AACAACCAGC
TTGTTGGTCG
2 01 CGGCACCAGC
GCCGTGGTCG
251 CGGATTATTA
GCCTAATAAT
3 01 GGCACGAAGT
CCGTGCTTCA
3 51 CGCTGTTTCC
GCGACAAAGG
4 01 GTGTGC CTGA
CACACGGACT
451 GGCAGATAGC
CCGTCTATCG
501 AACAAAGCAA
TTGTTTCGTT
551 GAGCAGTGGA
CTCGTCACCT
PCT/US01/15625
32/49
Figure 13 (cont)
SexAI
CCCAGCCGCC TTCAGTGAGT GGCGCACCAG GTCAGCGTGT
GGGTCGGCGG AAGTCACTCA CCGCGTGGTC CAGTCGCACA
TGTAGCGGCA GCAGCAGCAA CATTGGCAGC AACTATGTGA
ACATCGCCGT CGTCGTCGTT GTAACCGTCG TTGATACACT
Xmal
Smal
Aval
GCAGTTGCCC GGGACGGCGC CGAAACTGCT GATTTATGAT
CGTCAACGGG CCCTGCCGCG GCTTTGACGA CTAAATACTA
Bsu3 6I BamHI
GTCCCTCAGG CGTGCCGGAT CGTTTTAGCG GATCCAAAAG
CAGGGAGTCC GCACGGCCTA GCAAAATCGC CTAGGTTTTC
BpuAI
Bbsl
GCGAGCCTTG CGATTACGGG CCTGCAAAGC GAAGACGAAG
CGCTCGGAAC GCTAATGCCC GGACGTTTCG CTTCTGCTTC
Bsu.361
TTGCCAGAGC TATGACATGC CTCAGGCTGT GTTTGGCGGC
AACGGTCTCG ATACTGTACG GAGTCCGACA CAAACCGCCG
MscI Drain
TTAACCGTTC TTGGCCAGCC GAAAGCCGCA CCGAGTGTGA
AATTGGCAAG AACCGGTCGG CTTTCGGCGT GG C T C AC AC T
GCCGAGCAGC GAAGAATTGC AGGCGAACAA AGCGACCCTG
CGGCTCGTCG CTT CTTAACG TCCGCTTGTT TCGCTGGGAC
TTAGCGACTT TTATCCGGGA GCCGTGACAG TGGCCTGGAA
AAT CGCTGAA AATAGGCCCT CGGCACTGTC ACCGGACCTT
AGCCCCGTCA AGG CGGGAGT GGAGACCACC ACACCCTCCA
TCGGGGCAGT TCCGCCCTCA CCTCTGGTGG TGTGGGAGGT
CAACAAGTAC GCGGCCAGCA GCTATCTGAG CCTGACGCCT
GTTGTTCATG CGC CGGTCGT CGATAGACTC GGACTGCGGA
AGTCCCACAG AAGCTACAGC TGCCAGGTCA CGCATGAGGG
TCAGGGTGTC TTCGATGTCG ACGGTCCAGT GCGTACTCCC
StuI
Sphl
WO 01/87337 PCT/US01/15625
33/49
601 GAGCACCGTG GAAAAAACCG TTGCGCCGAC TGAGGCCTGA TAAGCATGCG
CTCGTGGCAC CTTTTTTGGC AACGCGGCTG ACTCCGGACT ATTCGTACGC
651 TAGGAGAAAA TAAAATGAAA CAAAGCACTA TTGCACTGGC ACTCTTACCG
ATCCTCTTTT ATTTTACTTT GTTTCGTGAT AACGTGACCG TGAGAATGGC
Mfel
701 TTGCTCTTCA CCCCTGTTAC CAAAGCCCAG GTGCAATTGA AAGAAAGCGG
AACGAGAAGT GGGGACAATG GTTTCGGGTC CACGTTAACT TTCTTTCGCC
BspEI
751 CCCGGCCCTG GTGAAAC CGA CCCAAACCCT GACCCTGACC TGTACCTTTT
GGGCCGGGAC CACTTTGGCT GGGTTTGGGA CTGGGACTGG ACATGGAAAA
BspEI
8 01 CCGGATTTAG CCTGTCCACG TCTGGCGTTG GCGTGGGCTG GATTCGCCAG
GGCCTAAATC GGACAGGTGC AGACCGCAAC CGCACCCGAC CTAAGCGGTC
Xhol
Aval
851 CCGCCTGGGA AAGCCCTCGA GTGGCTGGCT CTGATTGATT GGGATGATGA
GGCGGACCCT TTCGGGAGCT CACCGACCGA GAC TAACTAA CCCTACTACT
901 TAAGTATTAT AGCACCAGCC TGAAAACGCG TCTGACCATT AGCAAAGATA
ATTCATAATA TCGTGGTCGG ACTTTTGCGC AGACTGGTAA TCGTTTCTAT
BstBI
Sful
NspV
951 CTTCGAAAAA TCAGGTGGTG CTGACTATGA CCAACATGGA CCCGGTGGAT
GAAGCTTTTT AGTCCACCAC GACTGATACT GGTTGTACCT GGGCCACCTA
BssHII
1001 ACGGCCACCT ATTATTGCGC GCGTTCTCCT CGTTATCGTG GTGCTTTTGA
TGCCGGTGGA TAATAACGCG CGCAAGAGGA GCAATAGCAC CACGAAAACT
BlpI
Styl Celll
1051 TTATTGGGGC CAAGGCACCC TGGTGACGGT TAGCTCAGCG TCGACCAAAG
AATAACCCCG GTTCCGTGGG ACCACTGCCA ATCGAGTCGC AGCTGGTTTC
1101 GTCCAAGCGT GTTTCCGCTG GCTCCGAGCA GCAAAAGCAC CAGCGGCGGC
CAGGTTCGCA CAAAGGCGAC CGAGGCTCGT CGTTTTCGTG GTCGCCGCCG
1151 ACGGCTGCCC TGGGCTGCCT GGTTAAAGAT TATTTCCCGG AACCAGTCAC
WO 01/87337 PCT/US01/15625
34/49
TGCCGACGGG ACCCGACGGA CCAATTTCTA ATAAAGGGCC TTGGTCAGTG
12 01 CGTGAGCTGG AACAGCGGGG CGCTGACCAG CGGCGTGCAT ACCTTTCCGG
GCACTCGACC TTGTCGCCCC GCGACTGGTC GCCGCACGTA TGGAAAGGCC
1251 CGGTGCTGCA AAGCAGCGGC CTGTATAGCC TGAGCAGCGT TGTGACCGTG
GCCACGACGT TTCGTCGCCG GACATATCGG ACTCGTCGCA ACACTGGCAC
13 01 CCGAGCAGCA GCTTAGGCAC TCAGACCTAT ATTTGCAACG TGAAC CATAA
GGCTCGTCGT CGAATCCGTG AGT CTGGATA TAAACGTTGC ACTTGGTATT
EcoRI
13 51 ACCGAGCAAC AC CAAAGTGG ATAAAAAAGT GGAACCGAAA AGCGAATTCG
TGGCTCGTTG TGGTTTCACC TATTTTTTCA CCTTGGCTTT TCGCTTAAGC
BssHII
14 01 ACTATAAAGA TGACGATGAC AAAGGCGCGC CGTGGAGCCA CCCGCAGTTT
TGATATTTCT ACTGCTACTG TTTCCGCGCG GCACCTCGGT GGGCGT CAAA
Hindi I I
1451 GAAAAATGAT AAGCTTGACC TGTGAAGTGA AAAATGGCGC AGATTGTGCG
CTTTTTACTA TTCGAACTGG ACACTTCACT TTTTACCGCG TCTAACACGC
OGIII3 100.0%
1501 ACATTTTTTT TGTCTGCCGT TTAATTAAAG GGGGGGGGGG GCCGGCCTGG
TGTAAAAAAA ACAGACGGCA AATTAATTTC CCCCCCCCCC CGGCCGGACC
1551 GGGGGGGTGT ACATGAAATT GTAAACGTTA ATATTTTGTT AAAATTCGCG
CCCCCCCACA TGTACTTTAA CATTTGCAAT TATAAAACAA TTTTAAGCGC
1601 TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG
AATTTAAAAA CAATTTAGTC GAGTAAAAAA TTGGTTATCC GGCTTTAGCC
1651 CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG
GTTTTAGGGA ATATTTAGTT TTCTTATCTG GCTCTATCCC AACTCACAAC
1701 TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC
AAGGTCAAAC CTTGTTCTCA GGTGATAATT TCTTGCACCT GAGGTTGCAG
1751 AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC GAGAAC CAT C
TTTCCCGCTT TTTGGCAGAT AGTCCCGCTA CCGGGTGATG CTCTTGGTAG
18 01 ACCCTAATCA AGTTTTTTGG GGT CGAGGTG CCGTAAAGCA CTAAATCGGA
TGGGATTAGT TCAAAAAACC CCAGCTCCAC GGCATTTCGT GATTTAGCCT
1851 ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC
TGGGATTTCC CTCGGGGGCT AAATCTCGAA CTGCCCCTTT CGGCCGCTTG
1901 GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT
CACCGCTCTT TCCTTCCCTT CTTTCGCTTT CCTCGCCCGC GATCCCGCGA
1951 GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA
CCGTTCACAT CGCCAGTGCG ACGCGCATTG GTGGTGTGGG CGGCGCGAAT
WO 01/87337
35/49
PCT/US01/15625
2 001 ATGCGCCGCT ACAGGGCGCG TGCTAGACTA GTGTTTAAAC CGGACCGGGG
TACGCGGCGA TGTCCCGCGC ACGATCTGAT CACAAATTTG GCCTGGCCCC
2 051 GGGGGCTTAA GTGGGCTGCA AAACAAAACG GCCTCCTGTC AGGAAGCCGC
CCCCCGAATT CACCCGACGT TTTGTTTTGC CGGAGGACAG TCCTTCGGCG
2101 TTTTATCGGG TAGCCTCACT GCCCGCTTTC CAGTCGGGAA ACCTGTCGTG
AAAATAGCCC ATCGGAGTGA CGGGCGAAAG GTCAGCCCTT TGGACAGCAC
2151 CCAGCTGCAT CAGTGAATCG GCCAACGCGC GGGGAGAGGC GGTTTGCGTA
GGTCGACGTA GTCACTTAGC CGGTTGCGCG CCCCTCTCCG CCAAACGCAT
22 01 TTGGGAGCCA GGGTGGTTTT TCTTTTCACC AGTGAGACGG GCAACAGCTG
AACCCTCGGT CCCACCAAAA AGAAAAGTGG TCACTCTGCC CGTTGT CGAC
2251 ATTGCCCTTC ACCGCCTGGC CCTGAGAGAG TTGCAGCAAG CGGTCCACGC
TAACGGGAAG TGGCGGACCG GGACTCTCTC AACGTCGTTC GCCAGGTGCG
23 01 TGGTTTGCCC CAGCAGGCGA AAATCCTGTT TGATGGTGGT CAGCGGCGGG
ACCAAACGGG GTCGTCCGCT TTTAGGACAA ACTACCACCA GTCGCCGCCC
23 51 ATATAACATG AGCTGTCCTC GGTATCGTCG TATCCCACTA CCGAGATGTC
TATATTGTAC TCGACAGGAG CCATAGCAGC ATAGGGTGAT GGCTCTACAG
24 01 CGCACCAACG CGCAGCCCGG ACTCGGTAAT GGCACGCATT GCGCCCAGCG
GCGTGGTTGC GCGTCGGGCC TGAGC CATTA CCGTGCGTAA CGCGGGTCGC
2451 CCATCTGATC GTTGGCAACC AGCATCGCAG TGGGAACGAT GCCCTCATTC
GGTAGACTAG CAACCGTTGG TCGTAGCGTC ACCCTTGCTA CGGGAGTAAG
25 01 AGCATTTGCA TGGTTTGTTG AAAAC CGGAC ATGGCACTCC AGTCGCCTTC
TCGTAAACGT ACCAAACAAC TTTTGGCCTG TAC CGTGAGG TCAGCGGAAG
2551 CCGTTCCGCT ATCGGCTGAA TTTGATTGCG AGTGAGATAT TTATGCCAGC
GGCAAGGCGA TAGCCGACTT AAACTAACGC TCACTCTATA AATACGGTCG
2601 CAGC CAGACG CAGACGCGCC GAGACAGAAC TTAATGGGCC AGCTAACAGC
GTCGGTCTGC GTCTGCGCGG CTCTGTCTTG AATTACCCGG TCGATTGTCG
2651 GCGATTTGCT GGTGGCCCAA TGCGACCAGA TGCTCCACGC CCAGTCGCGT
CGCTAAACGA CCACCGGGTT ACGCTGGTCT ACGAGGTGCG GGTCAGCGCA
2 701 ACCGTCCTCA TGGGAGAAAA TAATACTGTT GATGGGTGTC TGGTCAGAGA
TGGCAGGAGT ACCCTCTTTT ATTATGACAA CTACCCACAG ACCAGTCTCT
2751 CAT C AAGAAA TAACGCCGGA ACATTAGTGC AGGCAGCTTC CACAGCAATA
GTAGTTCTTT ATTGCGGCCT TGTAATCACG TCCGTCGAAG GTGTCGTTAT
2 8 01 GCATCCTGGT CATCCAGCGG ATAGTTAATA ATCAGCCCAC TGACACGTTG
CGTAGGACCA GTAGGTCGCC TATCAATTAT TAGT CGGGTG ACTGTGCAAC
ApaLI
2 851 CGCGAGAAGA TTGTGCACCG CCGCTTTACA GGCTTCGACG CCGCTTCGTT
GCGCTCTTCT AACACGTGGC GGCGAAATGT CCGAAGCTGC GGCGAAGCAA
WO 01/87337
PCT/US01/15625
36/49
2 901 CTACCATCGA CACGACCACG CTGGCACCCA GTTGATCGGC GCGAGATTTA
GATGGTAGCT GTGCTGGTGC GACCGTGGGT CAACTAGCCG CGCTCTAAAT
2 951 ATCGCCGCGA CAATTTGCGA CGGCGCGTGC AGGGCCAGAC TGGAGGTGGC
TAGCGGCGCT GTTAAACGCT GCCGCGCACG TCCCGGTCTG ACCTCCACCG
3 001 AACGCCAATC AGCAACGACT GTTTGCCCGC CAGTTGTTGT GCCACGCGGT
TTGCGGTTAG TCGTTGCTGA CAAACGGGCG GTCAACAACA CGGTGCGCCA
3 051 TAGGAATGTA ATTCAGCTCC GCCATCGCCG CTTCCACTTT TTCCCGCGTT
ATCCTTACAT TAAGTCGAGG CGGTAGCGGC GAAGGTGAAA AAGGGCGCAA
3101 TTCGCAGAAA CGTGGCTGGC CTGGTTCACC ACGCGGGAAA CGGTCTGATA
AAGCGTCTTT GCACCGACCG GAC CAAGTGG TGCGCCCTTT GCCAGACTAT
3151 AGAGACACCG GCATACTCTG CGACAT CGTA TAACGTTACT GGTTTCACAT
TCTCTGTGGC CGTATGAGAC GCTGTAGCAT ATTGCAATGA CCAAAGTGTA
32 01 TCACCACCCT GAATTGACTC TCTTCCGGGC GCTATCATGC CATACCGCGA
AGTGGTGGGA CTTAACTGAG AGAAGGCCCG CGATAGTACG GTATGGCGCT
3 251 AAGGTTTTGC GCCATTCGAT GCTAGCCATG TGAGCAAAAG GCCAGCAAAA
TTCCAAAACG CGGTAAGCTA CGATCGGTAC ACTCGTTTTC CGGTCGTTTT
33 01 GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC
CCGGTCCTTG GCATTTTTCC GGCGCAACGA CCGCAAAAAG GTATCCGAGG
3 3 51 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA
CGGGGGGACT GCTCGTAGTG TTTTTAGCTG CGAGTTCAGT CTCCACCGCT
34 01 AACCCGACAG GAC T ATAAAG AT AC CAGGCG TTTCCCCCTG GAAGCTCCCT
TTGGGCTGTC CTGATATTTC TATGGTCCGC AAAGGGGGAC CTTCGAGGGA
3451 CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TAC CGGATAC CTGTCCGCCT
GCACGCGAGA GGACAAGGCT GGGACGGCGA ATGGCCTATG GACAGGCGGA
3501 TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT
AAGAGGGAAG CCCTTCGCAC CGCGAAAGAG TATCGAGTGC GACATC CATA
ApaLI
3 551 CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC
GAGTCAAGCC ACATCCAGCA AGCGAGGTTC GACCCGACAC ACGTGCTTGG
3 601 CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT
GGGGCAAGTC GGGCTGGCGA CGCGGAATAG GCCATTGATA GCAGAACTCA
3 651 CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC
GGTTGGGCCA TTCTGTGCTG AATAGCGGTG ACCGTCGTCG GTGACCATTG
3 701 AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG
TCCTAATCGT CTCGCTCCAT ACATCCGCCA CGATGT CTCA AGAACTTCAC
3 751 GTGGCCTAAC TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC
CAC CGGATTG ATGC CGATGT GATCTTCTTG TCATAAACCA TAGACGCGAG
3 8 01 TGCTGTAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC
WO 01/87337
PCT/US01/15625
37/49
ACGACATCGG TCAATGGAAG CCTTTTTCTC AACCATCGAG AACTAGGCCG
3 851 AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT
TTTGTTTGGT GGCGACCATC GCCACCAAAA AAACAAACGT TCGTCGTCTA
3901 TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG
ATGCGCGTCT TTTTTTCCTA GAGTTCTTCT AGGAAACTAG AAAAGATGCC
3 951 GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCAGA
CCAGACTGCG AGTCACCTTG CTTTTGAGTG CAATTCCCTA AAACCAGTCT
4 0 01 TCTAGCACCA GGCGTTTAAG GGCACCAATA ACTGCCTTAA AAAAATTACG
AGATCGTGGT CCGCAAATTC CCGTGGTTAT TGACGGAATT TTTTTAATGC
4 051 CCCCGCCCTG CCACTCATCG CAGTACTGTT GTAATTCATT AAGCATTCTG
GGGGCGGGAC GGTGAGTAGC GTCATGACAA CAT T AAGTAA TTCGTAAGAC
4101 CCGACATGGA AG C CAT C AC A AACGGCATGA TGAAC CTGAA TCGCCAGCGG
GGCTGTACCT TCGGTAGTGT TTGCCGTACT ACTTGGACTT AGCGGTCGCC
4151 CATCAGCACC TTGTCGCCTT GCGTATAATA TTTGCCCATA GTGAAAACGG
GTAGT CGTGG AACAGCGGAA CGCATATTAT AAACGGGTAT CACTTTTGCC
42 01 GGG CGAAGAA GTTGTCCATA TTGGCTACGT TTAAATCAAA ACTGGTGAAA
CCCGCTTCTT CAACAGGTAT AACCGATGCA AATTTAGTTT TGACCACTTT
4251 CTCACCCAGG GATTGGCTGA GACGAAAAAC ATATTCTCAA TAAACCCTTT
GAGTGGGTCC CTAACCGACT CTGCTTTTTG TAT AAGAGT T ATTTGGGAAA
43 01 AGGGAAATAG GCCAGGTTTT CACCGTAACA CGCCACATCT TGCGAATATA
TCCCTTTATC CGGTCCAAAA GTGGCATTGT GCGGTGTAGA ACGCTTATAT
43 51 TGTGTAGAAA CTGCCGGAAA TCGTCGTGGT ATTCACTCCA GAGCGATGAA
■ ACACATCTTT GACGGC CTTT AGCAGCACCA TAAGTGAGGT CTCGCTACTT
44 01 AACGTTTCAG TTTGCTCATG GAAAACGGTG TAACAAGGGT GAACACTATC
TTGCAAAGTC AAACGAGTAC CTTTTGCCAC ATTGTTCCCA CTTGTGATAG
44 51 CCATATCACC AGCTCACCGT CTTTCATTGC CATACGGAAC TCCGGGTGAG
GGTATAGTGG TCGAGTGGCA GAAAGTAACG GTATGCCTTG AGGCCCACTC
45 01 CATTCATCAG GCGGGCAAGA ATGTGAATAA AGGCCGGATA AAACTTGTGC
GTAAGTAGTC CGCCCGTTCT TACACTTATT TCCGGCCTAT TTTGAACACG
4551 TTATTTTTCT TTACGGTCTT TAAAAAGGCC GTAAT AT C C A GCTGAACGGT
AATAAAAAGA AATGCCAGAA ATTTTTCCGG CATTATAGGT CGACTTGCCA
46 01 CTGGTTATAG GTACATTGAG CAACTGACTG AAATGCCTCA AAATGTTCTT
GACCAATATC CATGTAACTC GTTGACTGAC TTTACGGAGT TTTACAAGAA
4651 TACGATGCCA TTGGGATATA TCAACGGTGG TATATCCAGT GATTTTTTTC
ATGCTACGGT AACCCTATAT AGTTGCCACC ATATAGGTCA CTAAAAAAAG
47 01 TCCATTTTAG CTTCCTTAGC TCCTGAAAAT CTCGATAACT CAAAAAATAC
AGGTAAAATC GAAGGAATCG AGGACTTTTA GAGCTATTGA GTTTTTTATG
4 751 GCCCGGTAGT GAT CTTATTT CATTATGGTG AAAGTTGGAA CCTCACCCGA
WO 01/87337
PCT/US01/15625
38/49
CGGGCCATCA CTAGAATAAA GTAATACCAC TTTCAACCTT GGAGTGGGCT
4 8 01 CGTCTAATGT GAGTTAGCTC ACTCATTAGG CACCCCAGGC TTTACACTTT
GCAGATTACA CTCAATCGAG TGAGTAATCC GTGGGGTCCG AAATGTGAAA
4851 ATGCTTCCGG CTCGTATGTT GTGTGGAATT GTGAGCGGAT AACAATTTCA
TACGAAGGCC GAGCATACAA CACACCTTAA CACTCGCCTA TTGTTAAAGT
Ml 3 Reverse primer 100.0% Xbal
4901 CACAGGAAAC AGCTATGACC ATGATTACGA ATTTCTAGAT AACGAGGGCA
GTGTCCTTTG TCGATACTGG TACTAATGCT TAAAGATCTA TTGCTCCCGT
4951 AAAAATGAAA AAGACAGCTA TCGCGATTGC AGTGGCACTG GCTGGTTTCG
TTTTTACTTT TTCTGTCGAT AGCGCTAACG TCACCGTGAC CGACCAAAGC
EcoRV
5 0 01 CTACCGTAGC GCAGGCCGAT
GATGGCATCG CGTCCGGCTA
WO 01/87337
PCT/US01/15625
39/49
Figure 14
M13 Reverse primer (100.0%)
\
SDSEQ
\\
LAC OPERATOR
MRNALAC OPERON \ \\
-ib.\ |
-35 \\ \
RV(87)
/ fee AI (123)
/ Acc 651 (191)
/ I Kpn 1(195)
//.XffKz I (205)
T->A
CdBExt2aigin
Apa LI (2803)
recognition stemloop
(252)
BamR I {211)
lbs I (335)
Bpu AI (335)
Bsu 361 (368)
Afrc 1(411)
Dra m (431)
5Vk I (722)
I (735)
e 1(821)
fsp EI (887)
Xho I (953)
va I (953)
A&p V(1040)
\sfu I (1040)
W BI (1040)
\Bss HH (1 104)
I (1147)
Orf 11(1171)
Blp 1(1171)
£coRI(1481)
'glllseqg (100.0%)
Cla I (1635)
glllss
A->C
C->A
ind m (1962)
OGII13 (100.0%)
WO 01/87337 PCT/US01/15625
40/49
Figure 14 (cont)
1 TCAGATAACG AGGGCAAAAA ATGAAAAAGA CAGCTATCGC GATTGCAGTG
AGTCTATTGC TCCCGTTTTT TACTTTTTCT GTCGATAGCG CTAACGTCAC
EcoRV
51 GCACTGGCTG GTTTCGCTAC CGTAGCGCAG GCCGATATCG TGCTGACCCA
CGTGACCGAC CAAAGCGATG GCATCGCGTC CGGCTATAGC ACGACTGGGT
SexAI
101 GCCGCCTTCA GTGAGTGGCG CACCAGGTCA GCGTGTGACC ATCTCGTGTA
CGGCGGAAGT CACTCACCGC GTGGTCCAGT CGCACACTGG TAGAGCACAT
Kpnl
Acc65I
151 GCGGCAGCAG CAG CAACATT GGCAGCAACT ATGTGAGCTG GTACCAGCAG
CGCCGTCGTC GTCGTTGTAA CCGTCGTTGA TACACTCGAC CATGGTCGTC
Xmal
Smal
Aval Bsu.3 61
201 TTGCCCGGGA CGGCGCCGAA ACTGCTGATT TATGATAACA ACCAGCGTCC
AACGGGCCCT GCCGCGGCTT TGACGACTAA ATACTATTGT TGGTCGCAGG
Bsu3 6I BamHI
251 CTCAGGCGTG CCGGATCGTT TTAGCGGATC CAAAAGCGGC ACCAGCGCGA
GAGTCCGCAC GGCCTAGCAA AA.T CGCCTAG GTTTTCGCCG TGGTCGCGCT
BpuAI
Bbsl
3 01 GCCTTGCGAT TACGGGCCTG CAAAGCGAAG ACGAAGCGGA TTATTATTGC
CGGAACGCTA ATGCCCGGAC GTTTCGCTTC TGCTTCGCCT AATAATAACG
Bsu.361
3 51 CAGAGCTATG ACATGCCTCA GGCTGTGTTT GGCGGCGGCA CGAAGTTTAA
GTCTCGATAC TGTACGGAGT CCGACACAAA CCGCCGCCGT GCTTCAAATT
MscI Dralll
4 01 CCGTTCTTGG CCAGCCGAAA GCCGCACCGA GTGTGACGCT GTTTCCGCCG
GGCAAGAACC GGTCGGCTTT CGGCGTGGCT CACACTGCGA CAAAGGCGGC
4 51 AGCAGCGAAG AATTGCAGGC GAACAAAGCG ACCCTGGTGT GCCTGATTAG
TCGTCGCTTC TTAACGTCCG CTTGTTTCGC TGGGACCACA CGGACTAATC
5 01 CGACTTTTAT CCGGGAGCCG TGACAGTGGC CTGGAAGGCA GATAGCAGCC
WO 01/87337
PCT/US01/15625
41/49
GCTGAAAATA GGCCCTCGGC ACTGTCACCG GACCTTCCGT CTATCGTCGG
551 CCGTCAAGGC GGGAGTGGAG ACCACCACAC CCTCCAAACA AAGCAACAAC
GGCAGTTCCG CCCTCACCTC TGGTGGTGTG GGAGGTTTGT TTCGTTGTTG
601 AAGTACGCGG CCAGCAGCTA TCTGAGCCTG ACGCCTGAGC AGTGGAAGTC
TTCATGCGCC GGTCGTCGAT AGACTCGGAC TGCGGACTCG TCACCTTCAG
651 CCACAGAAGC TACAGCTGCC AGGTCACGCA TGAGGGGAGC ACCGTGGAAA
GGTGTCTTCG ATGTCGACGG TCCAGTGCGT ACTCCCCTCG TGGCACCTTT
StuI SphI
701 AAACCGTTGC GCCGACTGAG GCCTGATAAG CATGCGTAGG AGAAAATAAA
TTTGGCAACG CGGCTGACTC CGGACTATTC GTACGCATCC TCTTTTATTT
751 ATGAAACAAA GCACTATTGC ACTGGCACTC TTACCGTTGC TCTTCACCCC
TACTTTGTTT CGTGATAACG TGAC CGTGAG AATGGCAACG AGAAGTGGGG
Mf el
8 01 TGTTACCAAA GCCCAGGTGC AATTGAAAGA AAGCGGCCCG GCCCTGGTGA
ACAATGGTTT CGGGTCCACG TTAACTTTCT TTCGCCGGGC CGGGAC CACT
BspEI
851 AACCGACCCA AACCCTGACC CTGACCTGTA CCTTTTCCGG ATTTAGCCTG
TTGGCTGGGT TTGGGACTGG GACTGGACAT GGAAAAGGCC TAAATCGGAC
901 TCCACGTCTG GCGTTGGCGT GGGCTGGATT CGCCAGCCGC CTGGGAAAGC
AGGTGCAGAC CGCAACCGCA CCCGACCTAA GCGGTCGGCG GACCCTTTCG
Xhol
Aval
951 CCTCGAGTGG CTGGCTCTGA TTGATTGGGA TGATGATAAG TATTATAGCA
GGAGCTCACC GAC CGAGACT AACTAACCCT ACTACTATTC ATAATATCGT
BstBI
Sful
NspV
10 01 CCAGCCTGAA AACGCGTCTG ' ACCATTAGCA AAGATACTTC GAAAAAT CAG
GGTCGGACTT TTGCGCAGAC TGGTAATCGT T T C TATGAAG CTTTTTAGTC
1051 GTGGTGCTGA CTATGACCAA CATGGACCCG GTGGATACGG CCACCTATTA
CACCACGACT GATACTGGTT GTACCTGGGC CACCTATGCC GGTGGATAAT
BssHII Styl
1101 TTGCGCGCGT TCTCCTCGTT ATCGTGGTGC TTTTGATTAT TGGGGCCAAG
AACGCGCGCA AGAGGAGCAA TAG C AC CACG AAAACTAATA ACCCCGGTTC
BlpI
WO 01/87337
42/49
PCT/US01/15625
Styl Celll
1151 GCACCCTGGT GACGGTTAGC TCAGCGTCGA CCAAAGGTCC AAGCGTGTTT
CGTGGGACCA CTGCCAATCG AGTCGCAGCT GGTTTCCAGG TTCGCACAAA
12 01 CCGCTGGCTC CGAGCAGCAA AAGCACCAGC GGCGGCACGG CTGCCCTGGG
GGCGACCGAG GCTCGTCGTT TTCGTGGTCG CCGCCGTGCC GACGGGAC C C
12 51 CTGCCTGGTT AAAGATTATT TCCCGGAACC AGTCACCGTG AGCTGGAACA
GACGGAC CAA TTTCTAATAA AGGGCCTTGG TCAGTGGCAC TCGACCTTGT
13 01 GCGGGGCGCT GAC CAGCGGC GTGCATACCT TTCCGGCGGT GCTGCAAAGC
CGCCCCGCGA CTGGTCGCCG CACGTATGGA AAGGCCGCCA CGACGTTTCG
13 51 AGCGGCCTGT ATAGC CTGAG CAGCGTTGTG ACCGTGCCGA GCAGCAGCTT
TCGCCGGACA TATCGGACTC GTCGCAACAC TGGCACGGCT CGTCGTCGAA
14 01 AGG C AC T C AG ACCTATATTT GCAACGTGAA CCATAAACCG AG C AACAC C A
TCCGTGAGTC TGGATATAAA CGTTGCACTT GGTATTTGGC TCGTTGTGGT
EcoRI
1451 AAGTGGATAA AAAAGTGGAA CCGAAAAGCG AATTCGGGGG AGGGAGCGGG
TTCACCTATT TTTTCACCTT GGCTTTTCGC TTAAGCCCCC TCCCTCGCCC
1501 AGCGGTGATT TTGATTATGA AAAGATGGCA AACGCTAATA AGGGGGCTAT
TCGCCACTAA AACTAATACT TTTCTACCGT TTGCGATTAT TCCCCCGATA
glllseq9 100.0%
1551 GACCGAAAAT GCCGATGAAA ACGCGCTACA GTCTGACGCT AAAGGCAAAC
CTGGCTTTTA CGGCTACTTT TGCGCGATGT CAGACTGCGA TTTCCGTTTG
Clal
16 01 TTGATTCTGT CGCTACTGAT TACGGTGCTG CTAT CGATGG TTTCATTGGT
AACTAAGACA GCGATGACTA ATGCCACGAC GATAGCTACC AAAGTAACCA
1651 GACGTTTCCG GCCTTGCTAA TGGTAATGGT GCTACTGGTG ATTTTGCTGG
CTGCAAAGGC CGGAACGATT AC CAT T AC C A CGATGAC CAC TAAAACGACC
1701 CTCTAATTCC CAAATGGCTC AAGTCGGTGA CGGTGATAAT TCACCTTTAA
GAGATTAAGG GTTTACCGAG TTCAGCCACT GCCACTATTA AGTGGAAATT
1751 TGAATAATTT CCGTCAATAT TTACCTTCCC TCCCTCAATC GGTTGAATGT
ACTTATTAAA GGCAGTTATA AATGGAAGGG AGGGAGTTAG CCAACTTACA
18 01 CGCCCTTTTG TCTTTGGCGC TGGTAAACCA TATGAATTTT CTATTGATTG
GCGGGAAAAC AGAAACCGCG ACCATTTGGT ATACTTAAAA GATAACTAAC
1851 TGACAAAATA AACTTATTCC GTGGTGTCTT TGCGTTTCTT TTATATGTTG
ACTGTTTTAT TTGAATAAGG CACCACAGAA ACGCAAAGAA AATATACAAC
1901 CCACCTTTAT GTATGTATTT TCTACGTTTG CTAACATACT GCGTAATAAG
GGTGGAAATA CATACATAAA AGATGCAAAC GATTGTATGA CGCATTATTC
WO 01/87337
43/49
Hindi I I
PCT/US01/15625
1951 GAGTCTTGAT AAGCTTGACC TGTGAAGTGA AAAATGGCGC AGATTGTGCG
CTCAGAACTA TTCGAACTGG ACACTTCACT TTTTACCGCG TCTAACACGC
OGIII3 100.0%
2001 ACATTTTTTT TGTCTGCCGT TTAATGAAAT TGTAAACGTT AATATTTTGT
TGTAAAAAAA ACAGACGGCA AATTACTTTA ACATTTGCAA TTATAAAACA
2 051 TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG
ATTTTAAGCG CAATTTAAAA ACAATTTAGT CGAGTAAAAA ATTGGTTATC
2101 GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG
CGGCTTTAGC CGTTTTAGGG AATATTTAGT TTTCTTATCT GGCTCTATCC
2151 GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG
CAACTCACAA CAAGGT CAAA CCTTGTTCTC AGGTGATAAT TTCTTGCACC
22 01 ACTCCAACGT CAAAGGGCGA AAAACCGTCT AT CAGGGCGA TGGCCCACTA
TGAGGTTGCA GTTTCCCGCT TTTTGGCAGA TAGTCCCGCT ACCGGGTGAT
2251 CGAGAAC CAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC
GCT CTTGGTA GTGGGATTAG TTCAAAAAAC CCCAGCTCCA CGGCATTTCG
23 01 ACTAAATCGG AAC C CTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA
TGATTTAGCC TTGGGATTTC CCTCGGGGGC TAAATCTCGA ACTGCCCCTT
23 51 AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAG CGAA AGGAGCGGGC
TCGGCCGCTT GCACCGCTCT TTCCTTCCCT TCTTTCGCTT TCCTCGCCCG
24 01 GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC
CGATCCCGCG ACCGTTCACA TCGCCAGTGC GACGCGCATT GGTGGTGTGG
2451 CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTGCTAGCCA TGTGAG CAAA
GCGGCGCGAA TTACGCGGCG ATGTCCCGCG CACGATCGGT ACACTCGTTT
2501 AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT
TCCGGTCGTT TTCCGGTCCT TGGCATTTTT CCGGCGCAAC GACCGCAAAA
2551 TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT
AGGTATCCGA GGCGGGGGGA CTGCTCGTAG TGTTTTTAGC TGCGAGTTCA
2601 CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC
GTCTCCACCG CTTTGGGCTG TCCTGATATT TCTATGGTCC GCAAAGGGGG
2651 TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT
ACCTTCGAGG GAGCACGCGA GAGGACAAGG CTGGGACGGC GAATGGCCTA
2701 ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA
TGGACAGGCG GAAAGAGGGA AGCCCTTCGC ACCGCGAAAG AGTAT CGAGT
2 751 CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG
GCGACATCCA TAGAGTCAAG CCACATCCAG CAAGCGAGGT TCGACCCGAC
ApaLI
WO 01/87337
PCT/US01/15625
44/49
28 01 TGTGCACGAA CCCCCCGTTC AGTCCGACCG CTGCGCCTTA TCCGGTAACT
ACACGTGCTT GGGGGGCAAG TCAGGCTGGC GACGCGGAAT AGGCCATTGA
2 851 ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA
TAGCAGAACT CAGGTTGGGC CATTCTGTGC TGAATAGCGG TGACCGTCGT
29 01 GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA
CGGTGACCAT TGTCCTAATC GTCTCGCTCC ATACATCCGC CACGATGTCT
2951 GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGA ACAGTATTTG
CAAGAACTTC AC CAC CGGAT TGATGC CGAT GTGATCTTCT TGT CATAAAC
3 001 GTATCTGCGC TCTGCTGTAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC
CATAGACGCG AGACGACATC GGT CAATGGA AGCCTTTTTC T CAAC CAT C G
3 051 TCTTGATCCG GCAAACAAAC CAC CGCTGGT AGCGGTGGTT TTTTTGTTTG
AGAACTAGGC CGTTTGTTTG GTGGCGACCA TCGCCACCAA AAAAACAAAC
3101 CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA
GTTCGTCGTC TAATGCGCGT CTTTTTTTCC TAGAGTTCTT CTAGGAAACT
3151 TCTTTTCTAC GGGGT CTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG
AGAAAAGATG CCCCAGACTG CGAGTCACCT TGCTTTTGAG TGCAATTCCC
32 01 ATTTTGGTCA GATCTAGCAC CAGGCGTTTA AGGGCAC CAA TAACTGCCTT
TAAAACCAGT CTAGATCGTG GTCCGCAAAT TCCCGTGGTT ATTGACGGAA
3251 AAAAAAATTA CGCCCCGCCC TGCCACTCAT CGCAGTACTG TTGTAATTCA
TTTTTTTAAT GCGGGGCGGG ACGGTGAGTA GCGTCATGAC AACATTAAGT
33 01 TTAAGCATTC TGCCGACATG G AAG C CAT C A CAAACGGCAT GATGAACCTG
AATT CGTAAG ACGGCTGTAC CTT CGGTAGT GTTTGCCGTA CTACTTGGAC
33 51 AATCGCCAGC GGCATCAGCA CCTTGTCGCC TTGCGTATAA TATTTGCCCA
TTAGCGGTCG CCGTAGTCGT GGAACAGCGG AACGCATATT ATAAACGGGT
34 01 TAGTGAAAAC GGGGGCGAAG AAGTTGTCCA TATTGGCTAC GTTTAAATCA
ATCACTTTTG CCCCCGCTTC TTCAACAGGT ATAAC CGATG CAAATTTAGT
3451 AAACTGGTGA AACTCACCCA GGGATTGGCT GAGACGAAAA ACATATTCTC
TTTGACCACT TTGAGTGGGT CCCTAACCGA CTCTGCTTTT TGTATAAGAG
3 5 01 AATAAACCCT TTAGGGAAAT AGGCCAGGTT TTCACCGTAA CACGCCACAT
TTATTTGGGA AATCCCTTTA TCCGGTCCAA AAGTGGCATT GTGCGGTGTA
3551 CTTGCGAATA TATGTGTAGA AACTGCCGGA AATCGTCGTG GTATTCACTC
GAACGCTTAT ATACACATCT TTGACGGCCT TTAGCAGCAC CATAAGTGAG
+ 1
3 601 CAGAGCGATG AAAACGTTTC AGTTTGCTCA TGGAAAACGG TGTAACAAGG
GTCTCGCTAC TTTTGCAAAG TCAAACGAGT ACCTTTTGCC ACATTGTTCC
3 651 GTGAACACTA TCCCATATCA CCAGCTCACC GTCTTTCATT GCCATACGGA
CACTTGTGAT AGGGTATAGT GGTCGAGTGG CAGAAAGTAA CGGTATGCCT
3 701 ACTCCGGGTG AGCATTCATC AGGCGGGCAA GAATGTGAAT AAAGGC CGGA
TGAGGCCCAC TCGTAAGTAG TCCGCCCGTT CTTACACTTA TTTCCGGCCT
WO 01/87337
45/49
PCT/US01/15625
3 751 TAAAACTTGT GCTTATTTTT CTTTACGGTC TTTAAAAAGG CCGTAATATC
ATTTTGAACA CGAATAAAAA GAAATGCCAG AAATTTTTCC GGCATTATAG
3 8 01 CAGCTGAACG GTCTGGTTAT AGGTACATTG AGCAACTGAC TGAAATGCCT
GTCGACTTGC CAGACCAATA TCCATGTAAC TCGTTGACTG ACTTTACGGA
3 851 CAAAATGTTC TTTACGATGC CATTGGGATA TATCAACGGT GGTATATCCA
GTTTTACAAG AAATGCTACG GTAACCCTAT ATAGTTGCCA CCATATAGGT
3 901 GTGATTTTTT TCTCCATTTT AGCTTCCTTA GCTCCTGAAA ATCTCGATAA
CACTAAAAAA AGAGGTAAAA TCGAAGGAAT CGAGGACTTT TAGAGCTATT
3 951 CTCAAAAAAT ACGCCCGGTA GTGATCTTAT TTCATTATGG TGAAAGTTGG
GAGTTTTTTA TGCGGGCCAT CACTAGAATA AAGTAATACC ACTTTCAACC
4001 AACCTCACCC GACGTCTAAT GTGAGTTAGC TCACTCATTA GGCACCCCAG
TTGGAGTGGG CTGCAGATTA CACTCAATCG AGTGAGTAAT CCGTGGGGTC
4 051 GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGGAA TTGTGAGCGG
CGAAATGTGA AATACGAAGG CCGAGCATAC AACACACCTT AACACTCGCC
Ml 3 Reverse primer 100.0%
4101 ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GAATT
TATTGTTAAA GTGTGTCCTT TGT CGATACT GGTACTAATG CTTAA
WO 01/87337 PCT/USOl/15625
46/49
Figure 15
MS-GPC-1:
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYY
C ARQ YG HRGGFDHWGQ G TL VT VS S
VL
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDFNE
SVFGGGTKLTVLG
MS-GPC-6
VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE
WVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCARGYGRYSPDLWGQGTLVTVSS
VL
DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLI
YGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYSNLPF
TFGQGTKVE I KRT
MS-GPC-8
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLALIDWDDDKYYSTSLKTRLTISKDTSKNQWLTMTNMDPVDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
WO 01/87337 PCT/USOl/15625
47/49
VL
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDMPQ
AVFGGGTKLTVLG
MS-GPC-10
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYY
CARQLHYRGGFDLWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDLTM
GVFGGGTKLTVLG
MS-GPC-8-6
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDYDH
YVFGGGTKLTVLG
MS-GPC-8-10
VH
WO 01/87337 PCT/USOl/15625
48/49
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLAL1DWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDLIRH
VFGGGTKLTVLG
MS-GPC-8-17
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLALIDWDDDKYYSTSLKTRLTISKDTSKNQWLTMTNMDPVDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDFSV
YVFG G GTKLTVLG
MS-GPC-8-27
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDMNV
HVFGG GTKLTVLG
WO 01/87337 PCT/USOl/15625
49/49
MS-GPC-8-6-13
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSESNIGANYVTWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDYDH
YVFGGGTKLTVLG
MS-GPC-8-10-57
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
W L AL I DWD D D KYYSTS LKTRLTI S KDTS KN Q VVLTMTN M D P VDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSESNIGNNYVQWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDLIRH
VFGGGTKLTVLG
MS-GPC-8-27-41
VH
QVQLKESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALE
WL AL I DWD D D KYYSTS L KTRLT I S KDTS KN QVVLTMTN M D P VDTATYY
CARSPRYRGAFDYWGQGTLVTVSS
VL
DIVLTQPPSVSGAPGQRVTISCSGSESNIGNNYVQWYQQLPGTAPKLLI
YDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSYDMNV
HVFGGGTKLTVLG
INTERNATIONAL SEARCH REPORT
Inte nal application No.
PC1V UE501/15625
A. CLASSIFICATION OF SUBJECT MATTER
IPC(7.)- :A6lK S9/S9S, 4.4
US CL :Please See Extra Sheet.
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
U.S. : 424/lSO.l, 133. 1, 138.1, 141.1, 143.1, 144.1, 152.1, 153. 1, 155.1, 172.1, 17S.1, 174.1
Documentation searched other than minimum documentation to the extent that such documents are included in the fields
searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
MEDLINE, BIOSIS, CANCERLIT, WEST
search terms antibodies, apoptosis, HLA-DR
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Cs
X
Y
X
Y
X
Y
x
* Special categories of cited documents:
"A" document defining the general state of the art which is not considered
to lie of particular relevance
"E" earlier document published on or after the international filing date
"L" document which may throw doubts on priority claun(s) or which is
cited to establish the publication date of another citation or other
special reason (as specified)
"0 M document referring to an oral disclosure, use, exhibition or other
means _
"F 1 document published prior to the international filing date but later
than the priority date claimed
Date of the actual completion of the international search
29 JULY 2001
Name and mailing address of the ISA/US
Commissioner of Patents and Trademarks
Box PCT
Washington, D.C. 20231
Facsimile No. (703) 305-3230
Relevant to claim No.
l
109, 110
1
109,110
1
109-110
"T"' later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
the prinoiple or theory underlying the invention
"X" document of particular relevance; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive step
when the dooument is taken alone
document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is combined
with one or more other such documents, such combination being
obvious to a person skilled in the art
"&" document member of the same patent family
Date of mailing of the international search report
15 AUG 208!
Authorized officer
KAREN A CANELLA
Telephone No. (703) 308-1235
tegory*
Citation of document, with indication, where appropriate, of the relevant passages
DUEYMES et al. Anti-endothelial cell antibody binding causes
apoptosis of endothelial cells. Arthritis & Rheumatism. September
1997, Vol.40, page S103. See abstract.
KIM et al. Altered expression of the genes regulating apoptosis in
multidrug resistant human myeloid leukemia cell lines overexpressing
MDR1 or MRP gene. 1997, Vol.11, pages 945-950. See abstract.
ISHIZUKA et al. Antitumour activity of murine monoclonal
antibody NCC-ST-421 on human cancer cells by inducing apoptosis.
Anticancer Research. July- August 1998, Vol.18, pages 2513-2518.
See absract.
Further documents are listed in the continuation of Box C. £ j See patent family annex.
Form PCT/ISA/210 (second sheet) (July 1998)*
INTERNATIONAL SEARCH REPORT
Intel nal application No.
PC1TUS01/1S625
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
X
Y
X
Y
X
Y
X
Y
X
Y
X
Y
X
Y
X
Y
C UT A H K .TCMANT et aL Activation of fas ligand/receptor system
kills ovarian cancer cell lines by apoptotic mechanism.
Gynecological Oncology. August 1998, Vol.70, pages 275-281. See
abstract
ILENSEL et aL Characterization of glycosylphosphatidylinositol-
lihked molecule CD55/decay- accelerating factor as the receptor for
antibody SC-l-induced apoptosis. Cancer Research. 15 October
1999, VoL59, pages 5299-5306. See abstract
NAKAMTIRA et aL Apoptosis induction of the human lung cancer
cell line in multicellular heterospheroids with human
antigangliosides GM2 monoclonal antibody. Cancer Research. 15
October 1999, Vol59, pages 5323-5330. See abstract
WAJJLiEN-OHMAN et aL Antibody- induced apoptosis in a human
leukemia cell line is energy dependent Cancer Letters. 10
December 1993, VoL75, pages 103-109. See abstract
MYSLER et aL The apoptosis- 1/Fas protein in human systmic
lupus erythematosus. Journal of Clinical Investigation. March
1994, VoL93, pages 1029-1034. See abstract
ACKERMAN et al. Induction of apoptotic or lytic cell death in
an ovarain adenocarcinoma cell line by antibodies generated
against a synthetic N-terminal extracellular domain gonadotropin-
releasing hormone receptor peptide. Cancer Letters. 30 June 1994,
Vol.81, pages 177-184. See abstract
ERAY et aL Cross- linking of surface IgG induces apoptosis in bcl-
2 expressing human follicular lymphoma line of mature B cell
phenotype. International Immunology. December 1994, Vol.6,
pages 1817-1827. See abstract
NAEAMTJRA et al. Apoptosis in human hepatoma cell line
induced by 4,5-didehydro geranylgeranoic acid via down- regulation
of transforming growth factor- alpha. Biochemical and Biophysical
Research Communications. 06 February 1996, Vol.219, pages 100-
104. See abstract
VOLLMERS et aL Apoptosis of stomach carcinoma cells induced
by a human monoclonal antibody. Cancer. 15 August 1995, Vol.76,
pages 550-558. See abstract
HATA et aL Fas/Apo-1 (CD95)-mediated and CD95- independent
apopto3ia of malignant plasma cclla. — Leukemia and Lymphoma.
1
109, 110
1
109,110
1
109, 110
1
109, 110
1
109, 110
1
109, 110
1
109, 110
1
109, 110
109, 110
¥ m PCT/IS Bem4 < ffeT il T§9o? V6r284? p^eV^-lt^ee abstract
109, 110
Inte nal apolication No.
INTERNATIONAL SEARCH REPORT
PClY USOl/15625
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
X
FOCtASJJ:IG AWA et al. FK506 inhibits anti-IgM antibody-
1
induced apoptosis and 17 kD endonuclease activity in the human
Y
B-cell line, MBC-1, established from BurkLtt's lymphoma. British
109, 110
Journal of Haematology, December 1997, Vol.99, pages 908-913.
See abstract.
x
MASTJDA et aL Dual action of CD30 antigen: anti-CD30 antibody
1
X
induced apoptosis and interleuMn-8 secretion in Ki-1 lymphoma
Y
cells. International Journal of Hematology, April 1998, Vol.67,
109, 110
pages 257-365. See abstract.
HAYAKAWA et al. A short peptide derived from the antisense
1
X
homology box of Fas ligand induces apoptosis in anti-Fas antibody-
"Y
A.
insensitive huamn ovarain cancer cells. Apoptosis. February 3000,
1AQ 11 A
XUy, XXV
VoL5, pages 37-41. See entire document.
■A
VEDOVIO et aL Selective apoptosis of neoplastic cells by the
1 c ly in 10 -in
1-0, <-iu, 10, ±y,
H 1 jA-DR-specific monoclonal antibody. Cancer Letters. 19 June
01
X
1998, V0LI88, pages 187-135. See entire document
6, 14, 15, 38, 66,
68, 69, 70, 109-
114.
x
LEE et aL H 1 A- PR-Triggered Inhibiton of Hemopoiesis Involves
1-5 7-10
Fas/Fas Ligand Interactions and Is Prevented by c-kit Ligand
6,
Y
Journal of Immunology. 01 October 1997, Vol.159, pages 3811-
14, 15, 38, 69 109-
3819. See entire document
1 1X
1X4
x
LEE et al HLA-DR- Mediated Signals for Hematopoiesis and
1 K ry -tf\
Induction of Apoptosis Involve But Are Not limited to a Nitric
Y
Oxide Pathway. Blood 01 July 1997, Vol.90, pages 817-225. See
6, 14, 15, 38, 69
entire document
1 no 1 1 x
ivy- 114
X
McDEVHT et al. Monoclonal anti-la antibody therapy in animal
1-5, 7-10, 66, 68,
models of autoimmune disease. Ciba Foundation Symp
osium.
70
Y
1987, Vol.129, pages 184-193. See entire document
fi 14 15 38 fiQ
U, J-Tj -Lis, *JO, \JX*
Y
HARRISON et al. Screening of Phage Antibody Libraries.
6, 69
Methods in Enzymology. 1996, Vol.267, pages 83-109.
See entire
document
Y
ROOS et aL Establishment and characterization of a human
14, 15
EBV- negative B cell line. Leukemia Research. 1988, V0L6, pages
685-693. See abstract
Form PCT/rSA/210 (continuation of second sheet) (July 1998)*
INTERNATIONAL SEARCH REPORT
Intel lal application No.
PC'i / U301/1562S
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
Y
TOSI et aL Immunochemical definition of the new dr specificity
8WJL)Ewl3. Immunological Communications. 1981, Vol.10, pages
275-893. See abstract
14, 15
Form PCT/ISA/210 (continuation of second sheet) (July 19S8)*
INTERNATIONAL SEARCH REPORT
Interr il application No.
PCT/USOl/15625
A. CLASSIFICATION OF SUBJECT MATTER:
US CL :
424/lSO.l, 138.1, 1SS.1, 141.1, 14S.1, 144.1, 1SS.1, 15S.1, 155.1, 172.1, 173.1, 174.1
Form PCT/rSA/210 (extra sheet) (July 1998)*
INTERNATIONAL SEARCH REPORT
Inter lal application No.
PCT/US01/1B625
Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)
This international report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:
□
Claims Nos.:
because they relate to subject matter not required to be searched by this Authority, namely:
□
Claims Nos.:
because they relate to parts of the international application that do not comply with the prescribed requirements to
such an extent that no meaningful international search can be carried out, specifically:
3. X Claims Nos.: 11-13,24-37,39-65,71-108
because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).
Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows:
1. |" | As all required additional search fees were timely paid by the applicant, this international search report covers all
searchable claims.
2. | | As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment
of any additional fee.
3. | | As only some of the required additional search fees were timely paid by the applicant, this international search report
covers only those claims for which fees were paid, specifically claims Nos.:
4. | | No required additional search fees were timely paid by the applicant. Consequently, this international search report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
Remark on Protest j The additional search fees were accompanied by the applicant's protest.
| | No protest accompanied the payment of additional search fees.
Form PCT/ISA/210 (continuation of first sheet(l)) (July 1998)*
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